Compositions, methods and kits for diagnosis of lung cancer

ABSTRACT

The present invention provides methods for identifying biomarker proteins that exhibit differential expression in subjects with a first lung condition versus healthy subjects or subjects with a second lung condition. The present invention also provides compositions comprising these biomarker proteins and methods of using these biomarker proteins or panels thereof to diagnose, classify, and monitor various lung conditions. The methods and compositions provided herein may be used to diagnose or classify a subject as having lung cancer or a non-cancerous condition, and to distinguish between different types of cancer (e.g., malignant versus benign, SCLC versus NSCLC).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Ser. No. 61/858,760, filed Jul. 26, 2013, the contents of which are hereby incorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “IDIA-009 Sequence listing_ST25.txt”, which was created on Sep. 29, 2014, and is 108 KB in size, are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Lung conditions and particularly lung cancer present significant diagnostic challenges. In many asymptomatic patients, radiological screens such as computed tomography (CT) scanning are a first step in the diagnostic paradigm. Pulmonary nodules (PNs) or indeterminate nodules are located in the lung and are often discovered during screening of both high risk patients or incidentally. The number of PNs identified is expected to rise due to increased numbers of patients with access to health care, the rapid adoption of screening techniques and an aging population. It is estimated that over 3 million PNs are identified annually in the US. Although the majority of PNs are benign, some are malignant leading to additional interventions. For patients considered low risk for malignant nodules, current medical practice dictates scans every three to six months for at least two years to monitor for lung cancer. The time period between identification of a PN and diagnosis is a time of medical surveillance or “watchful waiting” and may induce stress on the patient and lead to significant risk and expense due to repeated imaging studies. If a biopsy is performed on a patient who is found to have a benign nodule, the costs and potential for harm to the patient increase unnecessarily. Major surgery is indicated in order to excise a specimen for tissue biopsy and diagnosis. All of these procedures are associated with risk to the patient including: illness, injury and death as well as high economic costs.

Frequently, PNs cannot be biopsied to determine if they are benign or malignant due to their size and/or location in the lung. However, PNs are connected to the circulatory system, and so if malignant, protein markers of cancer can enter the blood and provide a signal for determining if a PN is malignant or not.

Diagnostic methods that can replace or complement current diagnostic methods for patients presenting with PNs are needed to improve diagnostics, reduce costs and minimize invasive procedures and complications to patients.

SUMMARY OF THE INVENTION

The present invention provides novel compositions, methods and kits for identifying protein markers to identify, diagnose, classify and monitor lung conditions, particularly lung cancer. The present invention uses a multiplexed assay to distinguish benign pulmonary nodules from malignant pulmonary nodules to classify patients with or without lung cancer. The present invention may be used in patients who present with symptoms of lung cancer, but do not have pulmonary nodules.

The present invention provides a method of determining the likelihood that a lung condition in a subject is cancer by measuring the abundance of proteins in a sample obtained from the subject; calculating a probability of cancer score based on the protein abundance and a protein-protein (mathematical) interaction between FRIL_HUMAN and COIA1_HUMAN; and ruling out cancer for the subject if the score is lower than a pre-determined score. When cancer is ruled out, the subject does not receive a treatment protocol. Treatment protocols include for example pulmonary function test (PFT), pulmonary imaging, a biopsy, a surgery, chemotherapy, a radiotherapy, or any combination thereof. In some embodiments, the imaging is an x-ray, a chest computed tomography (CT) scan, or a positron emission tomography (PET) scan.

The present invention further provides a method of determining the likelihood of the presence of a lung condition in a subject by measuring the abundance of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein abundance and a protein-protein (mathematical) interaction between FRIL_HUMAN and COIA1_HUMAN; and concluding the presence of said lung condition if the score is equal or greater than a pre-determined score. The pre-determined score can be determined by scoring a plurality of subjects as part of a reference population. The lung condition is lung cancer such as for example, non-small cell lung cancer (NSCLC). The subject is at risk of developing lung cancer. The likelihood of cancer can be determined by the sensitivity, specificity, negative predictive value or positive predictive value associated with the score.

The present invention also provides methods of determining that a lung condition in a subject is cancer comprising assessing the expression of a plurality of proteins comprising determining the protein expression level of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN from a biological sample obtained from the subject; calculating a score from the protein expression of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN from the biological sample determined in the preceding step; and comparing the score from the biological sample to a plurality of scores obtained from a reference population, wherein the comparison provides a determination that the lung condition is not cancer.

The determination that a lung condition is not cancer can include assessing the expression of a plurality of proteins to determine the protein expression level of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN obtained from a biological sample from a subject. A score is calculated from these assessments and this score is further compared with a plurality of scores obtained from a reference population, wherein the comparison provides a determination that the lung condition is not cancer. The method can also include determining an interaction between FRIL_HUMAN AND COIA1_HUMAN.

Comparing the score from the subject with the plurality of scores obtained from the reference population can provide a cancer probability. Preferably, when the comparison provides a cancer probability and the probability is 15% or less, the lung condition is classified as not cancer. More preferably, when the comparison provides a cancer probability and the probability is 10% or less, the lung condition is classified as not cancer. Most preferably, when the comparison provides a cancer probability and the probability is 5% or less, the lung condition is classified as not cancer.

The subject can be one that has or is suspected of having a pulmonary nodule. The pulmonary nodule can have a diameter of 30 mm or less. Preferably, the pulmonary nodule has a diameter of about 8 mm to 30 mm.

The subject can be suspected of having a cancerous or non-cancerous lung condition. A cancerous lung condition can include non-small cell lung cancer. A non-cancerous lung condition can include chronic obstructive pulmonary disease, hamartoma, fibroma, neurofibroma, granuloma, sarcoidosis, bacterial infection or fungal infection.

The subject can be a mammal. Preferably, the subject is a human.

The biological sample can be any sample obtained from the subject, e.g., tissue, cell, fluid. Preferably, the biological sample is tissue, blood, plasma, serum, whole blood, urine, saliva, genital secretions, cerebrospinal fluid, sweat, excreta, or bronchoalveolar lavage.

The methods of the present invention can also include assessing the expression of a plurality of proteins which comprises determining the protein expression level of at least one of PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN and PTPRJ_HUMAN.

Determining the protein level of at least one of, or each of, the proteins of the present invention can include fragmenting the protein to generate at least one peptide per protein. Preferably, the fragmentation of the protein is accomplished by trypsin digestion.

The methods of the present invention can further include normalizing the protein measurements. For example, the protein measurements can normalized by one or more “housekeeping” proteins, e.g., proteins which do not have variable expression across different samples or subjects. Preferable normalizing proteins can include at least one of PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN and PTPRJ_HUMAN.

The invention further provides methods of using synthetic, modified, heavy peptides corresponding to at least one of, or each of, ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, COIA1_HUMAN, PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN or PTPRJ_HUMAN. At least one of, or each of, the synthetic peptides can an isotopic label attached.

Methods to assess the expression of a plurality of proteins can include mass spectrometry (MS), liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM-MS), reverse transcriptase-polymerase chain reaction (RT-PCR), microarray, serial analysis of gene expression (SAGE), gene expression analysis by massively parallel signature sequencing (MPSS), immunoassays, immunohistochemistry (IHC), transcriptomics, or proteomics. Preferably, the expression of a plurality of proteins is assessed LC-SRM-MS. LC-SRM-MS can be used to determine transitions for each peptide analyzed. Preferably, peptide transitions can be determined for at least one of, or each of, ALQASALK (SEQ ID NO: 25), AVGLAGTFR (SEQ ID NO: 26), GFLLLASLR (SEQ ID NO: 27), LGGPEAGLGEYLFER (SEQ ID NO: 28) or VEIFYR (SEQ ID NO: 29). More preferably the peptide transitions include at least ALQASALK (SEQ ID NO: 25) (401.25, 617.4), AVGLAGTFR (SEQ ID NO: 26) (446.26, 721.4), GFLLLASLR (SEQ ID NO: 27) (495.31, 559.4), LGGPEAGLGEYLFER (SEQ ID NO: 28) (804.4, 1083.6), and VEIFYR (SEQ ID NO: 29) (413.73, 598.3).

The measuring step may also be performed using a compound that specifically binds the protein being detected or a peptide transition. For example, a compound that specifically binds to the protein being measured can be an antibody or an aptamer.

The score can be calculated from a logistic regression model applied to the protein measurements. For example, the score is determined as P_(s)=1/[1+exp(−α−Σ_(i=1) ⁵β_(i)*{hacek over (I)}_(i,s)−γ*{hacek over (I)}_(COIA1)*{hacek over (I)}_(FRIL))], where {hacek over (I)}_(i,s) is Box-Cox transformed and normalized intensity of transition i in said sample (s), β_(i) is the corresponding logistic regression coefficient, α is a panel-specific constant, and γ is a coefficient for the interaction term.

The reference population can include at least 100 subjects with a lung condition and wherein each subject in the reference population has been assigned a score based on the protein expression of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN obtained from a biological sample from the subject. The invention further provides methods for the treatment of a subject, wherein if the lung condition is not cancer the subject is treated based on clinical practice guidelines. Preferably, if a lung condition is not cancer the subject receives image monitoring for at least a 1 year period, for at least a 2 year period or at least a 3 year period. More preferably, if the lung condition is not cancer, the subject receives chest computed tomography scans for at least a 1 year period, for at least a 2 year period or at least a 3 year period.

The present invention also provides that at least one step of any disclosed method can be performed on a computer or computer system.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. GenBank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this disclosure has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the disclosure encompassed by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing sample batches used in the experiments from three sites UPenn, Laval and NYU.

FIG. 2 is a panel of graphs showing A) NPV and specificity of panel ID_(—)465 and B) area under the curve for a receiving operating curve for panel ID_(—)465.

FIG. 3 is a panel of graphs showing A) NPV and specificity of panel ID_(—)341 and B) area under the curve for a receiving operating curve for panel ID_(—)341.

FIG. 4 is a graph showing NPV and specificity of panel ID_(—)465 plus COIA1×FRIL interaction (C4 Classifier).

FIG. 5 is a graph showing NPV and specificity of panel ID_(—)341.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed invention derives from the surprising discovery, that in patients presenting with pulmonary nodule(s), a small panel of protein markers in the blood is able to specifically identify and distinguish malignant and benign lung nodules with high negative predictive value (NPV). More importantly, at least two protein markers among the panel mathematically interact in the model for determining the probability score. Such protein-protein interaction surprisingly increases the specificity of the methods described herein. The classifier (C4 Classifier) described herein also demonstrates remarkable independence and accuracy. None of the clinical factors impact the classifier's score.

Accordingly the invention provides unique advantages to the patient associated with early detection of lung cancer in a patient, including increased life span, decreased morbidity and mortality, decreased exposure to radiation during screening and repeat screenings and a minimally invasive diagnostic model. Importantly, the methods of the invention allow for a patient to avoid invasive procedures.

The routine clinical use of chest computed tomography (CT) scans identifies millions of pulmonary nodules annually, of which only a small minority are malignant but contribute to the dismal 15% five-year survival rate for patients diagnosed with non-small cell lung cancer (NSCLC). The early diagnosis of lung cancer in patients with pulmonary nodules is a top priority, as decision-making based on clinical presentation, in conjunction with current non-invasive diagnostic options such as chest CT and positron emission tomography (PET) scans, and other invasive alternatives, has not altered the clinical outcomes of patients with Stage I NSCLC. The subgroup of pulmonary nodules between 8 mm and 20 mm in size is increasingly recognized as being “intermediate” relative to the lower rate of malignancies below 8 mm and the higher rate of malignancies above 20 mm. Invasive sampling of the lung nodule by biopsy using transthoracic needle aspiration or bronchoscopy may provide a cytopathologic diagnosis of NSCLC, but are also associated with both false-negative and non-diagnostic results. In summary, a key unmet clinical need for the management of pulmonary nodules is a non-invasive diagnostic test that discriminates between malignant and benign processes in patients with indeterminate pulmonary nodules (IPNs), especially between 8 mm and 20 mm in size.

The clinical decision to be more or less aggressive in treatment is based on risk factors, primarily nodule size, smoking history and age in addition to imaging. As these are not conclusive, there is a great need for a molecular-based blood test that would be both non-invasive and provide complementary information to risk factors and imaging.

Accordingly, these and related embodiments will find uses in screening methods for lung conditions, and particularly lung cancer diagnostics. More importantly, the invention finds use in determining the clinical management of a patient. That is, the method of invention is useful in ruling in or ruling out a particular treatment protocol for an individual subject.

Cancer biology requires a molecular strategy to address the unmet medical need for an assessment of lung cancer risk. The field of diagnostic medicine has evolved with technology and assays that provide sensitive mechanisms for detection of changes in proteins. The methods described herein use a LC-SRM-MS technology for measuring the concentration of blood plasma proteins that are collectively changed in patients with a malignant PN. This protein signature is indicative of lung cancer. LC-SRM-MS is one method that provides for both quantification and identification of circulating proteins in plasma. Changes in protein expression levels, such as but not limited to signaling factors, growth factors, cleaved surface proteins and secreted proteins, can be detected using such a sensitive technology to assay cancer. Presented herein is a blood-based classification test to determine the likelihood that a patient presenting with a pulmonary nodule has a nodule that is benign or malignant. The present invention presents a classification algorithm that predicts the relative likelihood of the PN being benign or malignant.

More broadly, it is demonstrated that there are many variations on this invention that are also diagnostic tests for the likelihood that a PN is benign or malignant. These are variations on the panel of proteins, protein standards, measurement methodology and/or classification algorithm.

The present invention also provides methods of determining that a lung condition in a subject is cancer comprising assessing the expression of a plurality of proteins comprising determining the protein expression level of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN from a biological sample obtained from the subject; calculating a score from the protein expression of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN from the biological sample determined in the preceding step; and comparing the score from the biological sample to a plurality of scores obtained from a reference population, wherein the comparison provides a determination that the lung condition is not cancer.

The determination that a lung condition is not cancer can include assessing the expression of a plurality of proteins to determine the protein expression level of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN obtained from a biological sample from a subject. A score is calculated from these assessments and this score is further compared with a plurality of scores obtained from a reference population, wherein the comparison provides a determination that the lung condition is not cancer. The method can also include determining an interaction between FRIL_HUMAN AND COIA1_HUMAN.

Comparing the score from the subject with the plurality of scores obtained from the reference population can provide a cancer probability. Preferably, when the comparison provides a cancer probability and the probability is 15% or less, the lung condition is classified as not cancer. More preferably, when the comparison provides a cancer probability and the probability is 10% or less, the lung condition is classified as not cancer. Most preferably, when the comparison provides a cancer probability and the probability is 5% or less, the lung condition is classified as not cancer.

The subject can be one that has or is suspected of having a pulmonary nodule. The pulmonary nodule can have a diameter of 30 mm or less. Preferably, the pulmonary nodule has a diameter of about 8 mm to 30 mm.

The subject can be suspected of having a cancerous or non-cancerous lung condition. A cancerous lung condition can include non-small cell lung cancer. A non-cancerous lung condition can include chronic obstructive pulmonary disease, hamartoma, fibroma, neurofibroma, granuloma, sarcoidosis, bacterial infection or fungal infection.

The subject can be a mammal. Preferably, the subject is a human.

The biological sample can be any sample obtained from the subject, e.g., tissue, cell, fluid. Preferably, the biological sample is tissue, blood, plasma, serum, whole blood, urine, saliva, genital secretions, cerebrospinal fluid, sweat, excreta, or bronchoalveolar lavage.

The methods of the present invention can also include assessing the expression of a plurality of proteins which comprises determining the protein expression level of at least one of PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN and PTPRJ_HUMAN.

Determining the protein level of at least one of, or each of, the proteins of the present invention can include fragmenting the protein to generate at least one peptide per protein. Preferably, the fragmentation of the protein is accomplished by trypsin digestion.

The methods of the present invention can further include normalizing the protein measurements. For example, the protein measurements can normalized by one or more “housekeeping” proteins, e.g., proteins which do not have variable expression across different samples or subjects. Preferable normalizing proteins can include at least one of PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN and PTPRJ_HUMAN.

The invention further provides methods of using synthetic, modified, heavy peptides corresponding to at least one of, or each of, ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, COIA1_HUMAN, PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN or PTPRJ_HUMAN. At least one of, or each of, the synthetic peptides can an isotopic label attached.

Methods to assess the expression of a plurality of proteins can include mass spectrometry (MS), liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM-MS), reverse transcriptase-polymerase chain reaction (RT-PCR), microarray, serial analysis of gene expression (SAGE), gene expression analysis by massively parallel signature sequencing (MPSS), immunoassays, immunohistochemistry (IHC), transcriptomics, or proteomics. Preferably, the expression of a plurality of proteins is assessed LC-SRM-MS. LC-SRM-MS can be used to determine transitions for each peptide analyzed. Preferably, peptide transitions can be determined for at least one of, or each of, ALQASALK (SEQ ID NO: 25), AVGLAGTFR (SEQ ID NO: 26), GFLLLASLR (SEQ ID NO: 27), LGGPEAGLGEYLFER (SEQ ID NO: 28) or VEIFYR (SEQ ID NO: 29). More preferably the peptide transitions include at least ALQASALK (SEQ ID NO: 25) (401.25, 617.4), AVGLAGTFR (SEQ ID NO: 26) (446.26, 721.4), GFLLLASLR (SEQ ID NO: 27) (495.31, 559.4), LGGPEAGLGEYLFER (SEQ ID NO: 28) (804.4, 1083.6), and VEIFYR (SEQ ID NO: 29) (413.73, 598.3).

The measuring step may also be performed using a compound that specifically binds the protein being detected or a peptide transition. For example, a compound that specifically binds to the protein being measured can be an antibody or an aptamer.

The score can be calculated from a logistic regression model applied to the protein measurements. For example, the score is determined as P_(s)=1/[1+exp(−α−Σ_(i=1) ⁵β_(i)*{hacek over (I)}_(i,s)−γ*{hacek over (I)}_(COIA1)*{hacek over (I)}_(FRIL))], where {hacek over (I)}_(i,s) is Box-Cox transformed and normalized intensity of transition i in said sample (s), β_(i) is the corresponding logistic regression coefficient, α is a panel-specific constant, and γ is a coefficient for the interaction term.

The reference population can include at least 100 subjects with a lung condition and wherein each subject in the reference population has been assigned a score based on the protein expression of at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN obtained from a biological sample from the subject. The invention further provides methods for the treatment of a subject, wherein if the lung condition is not cancer the subject is treated based on clinical practice guidelines. Preferably, if a lung condition is not cancer the subject receives image monitoring for at least a 1 year period, for at least a 2 year period or at least a 3 year period. More preferably, if the lung condition is not cancer, the subject receives chest computed tomography scans for at least a 1 year period, for at least a 2 year period or at least a 3 year period.

The present invention also provides that at least one step of any disclosed method can be performed on a computer or computer system.

As disclosed herein, archival plasma samples from subjects presenting with PNs were analyzed for differential protein expression by mass spectrometry and the results were used to identify biomarker proteins and panels of biomarker proteins that are differentially expressed in conjunction with various lung conditions (cancer vs. non-cancer).

In one aspect of the invention, the panel comprises at least 2, 3, 4, 5, or more protein markers with at least one protein-protein interaction. In some embodiments, the panel comprises 5 protein markers with at least one protein-protein interaction. In some embodiments, the panel comprises ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN; and FRIL_HUMAN and COIA1_HUMAN interact in the model for determining the probability score of cancer. In some embodiments, the panel comprises 2, 3, or 4 biomarkers selected from the group consisting of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN; and at least one protein-protein mathematical interaction exits among the biomarkers.

Additional biomarkers that can be used herein are described in WO 13/096845, the contents of which are incorporated herein by reference in its entireties.

The term “interact”, “interacted”, “interaction” or “protein-protein interaction” used herein refers to mathematical interaction between peptides (or peptide transitions) derived from two or more protein markers when calculating the probability score of cancer.

The term “pulmonary nodules” (PNs) refers to lung lesions that can be visualized by radiographic techniques. A pulmonary nodule is any nodules less than or equal to three centimeters in diameter. In one example a pulmonary nodule has a diameter of about 0.8 cm to 2 cm.

The term “masses” or “pulmonary masses” refers to lung nodules that are greater than three centimeters maximal diameter.

The term “blood biopsy” refers to a diagnostic study of the blood to determine whether a patient presenting with a nodule has a condition that may be classified as either benign or malignant.

The term “acceptance criteria” refers to the set of criteria to which an assay, test, diagnostic or product should conform to be considered acceptable for its intended use. As used herein, acceptance criteria are a list of tests, references to analytical procedures, and appropriate measures, which are defined for an assay or product that will be used in a diagnostic. For example, the acceptance criteria for the classifier refer to a set of predetermined ranges of coefficients.

The term “average maximal AUC” refers to the methodology of calculating performance. For the present invention, in the process of defining the set of proteins that should be in a panel by forward or backwards selection proteins are removed or added one at a time. A plot can be generated with performance (AUC or partial AUC score on the Y axis and proteins on the X axis) the point which maximizes performance indicates the number and set of proteins the gives the best result.

The term “partial AUC factor or pAUC factor” is greater than expected by random prediction. At sensitivity=0.90 the pAUC factor is the trapezoidal area under the ROC curve from 0.9 to 1.0 Specificity/(0.1*0.1/2).

The term “incremental information” refers to information that may be used with other diagnostic information to enhance diagnostic accuracy. Incremental information is independent of clinical factors such as including nodule size, age, or gender.

The term “score” or “scoring” refers to calculating a probability likelihood for a sample. For the present invention, values closer to 1.0 are used to represent the likelihood that a sample is cancer, values closer to 0.0 represent the likelihood that a sample is benign.

The term “robust” refers to a test or procedure that is not seriously disturbed by violations of the assumptions on which it is based. For the present invention, a robust test is a test wherein the proteins or transitions of the mass spectrometry chromatograms have been manually reviewed and are “generally” free of interfering signals.

The term “coefficients” refers to the weight assigned to each protein used to in the logistic regression model to score a sample.

In certain embodiments of the invention, it is contemplated that in terms of the logistic regression model of MC CV, the model coefficient and the coefficient of variation (CV) of each protein's model coefficient may increase or decrease, dependent upon the method (or model) of measurement of the protein classifier. For each of the listed proteins in the panels, there is about, at least, at least about, or at most about a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-, -fold or any range derivable therein for each of the coefficient and CV. Alternatively, it is contemplated that quantitative embodiments of the invention may be discussed in terms of as about, at least, at least about, or at most about 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more, or any range derivable therein.

The term “best team players” refers to the proteins that rank the best in the random panel selection algorithm, i.e., perform well on panels. When combined into a classifier these proteins can segregate cancer from benign samples. “Best team player proteins” are synonymous with “cooperative proteins”. The term “cooperative proteins” refers to proteins that appear more frequently on high performing panels of proteins than expected by chance. This gives rise to a protein's cooperative score which measures how (in) frequently it appears on high performing panels. For example, a protein with a cooperative score of 1.5 appears on high performing panels 1.5× more than would be expected by chance alone.

The term “classifying” as used herein with regard to a lung condition refers to the act of compiling and analyzing expression data for using statistical techniques to provide a classification to aid in diagnosis of a lung condition, particularly lung cancer.

The term “classifier” as used herein refers to an algorithm that discriminates between disease states with a predetermined level of statistical significance. A two-class classifier is an algorithm that uses data points from measurements from a sample and classifies the data into one of two groups. In certain embodiments, the data used in the classifier is the relative expression of proteins in a biological sample. Protein expression levels in a subject can be compared to levels in patients previously diagnosed as disease free or with a specified condition. Table 4 lists a representative classifier (C4 Classifier).

The “classifier” maximizes the probability of distinguishing a randomly selected cancer sample from a randomly selected benign sample, i.e., the AUC of ROC curve.

In addition to the classifier's constituent proteins with differential expression, it may also include proteins with minimal or no biologic variation to enable assessment of variability, or the lack thereof, within or between clinical specimens; these proteins may be termed endogenous proteins and serve as internal controls for the other classifier proteins.

The term “normalization” or “normalizer” as used herein refers to the expression of a differential value in terms of a standard value to adjust for effects which arise from technical variation due to sample handling, sample preparation and mass spectrometry measurement rather than biological variation of protein concentration in a sample. For example, when measuring the expression of a differentially expressed protein, the absolute value for the expression of the protein can be expressed in terms of an absolute value for the expression of a standard protein that is substantially constant in expression. This prevents the technical variation of sample preparation and mass spectrometry measurement from impeding the measurement of protein concentration levels in the sample.

The term “condition” as used herein refers generally to a disease, event, or change in health status.

The term “treatment protocol” as used herein including further diagnostic testing typically performed to determine whether a pulmonary nodule is benign or malignant. Treatment protocols include diagnostic tests typically used to diagnose pulmonary nodules or masses such as for example, CT scan, positron emission tomography (PET) scan, bronchoscopy or tissue biopsy. Treatment protocol as used herein is also meant to include therapeutic treatments typically used to treat malignant pulmonary nodules and/or lung cancer such as for example, chemotherapy, radiation or surgery.

The terms “diagnosis” and “diagnostics” also encompass the terms “prognosis” and “prognostics”, respectively, as well as the applications of such procedures over two or more time points to monitor the diagnosis and/or prognosis over time, and statistical modeling based thereupon. Furthermore the term diagnosis includes: a. prediction (determining if a patient will likely develop a hyperproliferative disease) b. prognosis (predicting whether a patient will likely have a better or worse outcome at a pre-selected time in the future) c. therapy selection d. therapeutic drug monitoring e. relapse monitoring.

In some embodiments, for example, classification of a biological sample as being derived from a subject with a lung condition may refer to the results and related reports generated by a laboratory, while diagnosis may refer to the act of a medical professional in using the classification to identify or verify the lung condition.

The term “providing” as used herein with regard to a biological sample refers to directly or indirectly obtaining the biological sample from a subject. For example, “providing” may refer to the act of directly obtaining the biological sample from a subject (e.g., by a blood draw, tissue biopsy, lavage and the like). Likewise, “providing” may refer to the act of indirectly obtaining the biological sample. For example, providing may refer to the act of a laboratory receiving the sample from the party that directly obtained the sample, or to the act of obtaining the sample from an archive.

As used herein, “lung cancer” preferably refers to cancers of the lung, but may include any disease or other disorder of the respiratory system of a human or other mammal. Respiratory neoplastic disorders include, for example small cell carcinoma or small cell lung cancer (SCLC), non-small cell carcinoma or non-small cell lung cancer (NSCLC), squamous cell carcinoma, adenocarcinoma, broncho-alveolar carcinoma, mixed pulmonary carcinoma, malignant pleural mesothelioma, undifferentiated large cell carcinoma, giant cell carcinoma, synchronous tumors, large cell neuroendocrine carcinoma, adenosquamous carcinoma, undifferentiated carcinoma; and small cell carcinoma, including oat cell cancer, mixed small cell/large cell carcinoma, and combined small cell carcinoma; as well as adenoid cystic carcinoma, hamartomas, mucoepidermoid tumors, typical carcinoid lung tumors, atypical carcinoid lung tumors, peripheral carcinoid lung tumors, central carcinoid lung tumors, pleural mesotheliomas, and undifferentiated pulmonary carcinoma and cancers that originate outside the lungs such as secondary cancers that have metastasized to the lungs from other parts of the body. Lung cancers may be of any stage or grade. Preferably the term may be used to refer collectively to any dysplasia, hyperplasia, neoplasia, or metastasis in which the protein biomarkers expressed above normal levels as may be determined, for example, by comparison to adjacent healthy tissue.

Examples of non-cancerous lung condition include chronic obstructive pulmonary disease (COPD), benign tumors or masses of cells (e.g., hamartoma, fibroma, neurofibroma), granuloma, sarcoidosis, and infections caused by bacterial (e.g., tuberculosis) or fungal (e.g. histoplasmosis) pathogens. In certain embodiments, a lung condition may be associated with the appearance of radiographic PNs.

As used herein, “lung tissue”, and “lung cancer” refer to tissue or cancer, respectively, of the lungs themselves, as well as the tissue adjacent to and/or within the strata underlying the lungs and supporting structures such as the pleura, intercostal muscles, ribs, and other elements of the respiratory system. The respiratory system itself is taken in this context as representing nasal cavity, sinuses, pharynx, larynx, trachea, bronchi, lungs, lung lobes, aveoli, aveolar ducts, aveolar sacs, aveolar capillaries, bronchioles, respiratory bronchioles, visceral pleura, parietal pleura, pleural cavity, diaphragm, epiglottis, adenoids, tonsils, mouth and tongue, Substitute Specification-Clean Copy and the like. The tissue or cancer may be from a mammal and is preferably from a human, although monkeys, apes, cats, dogs, cows, horses and rabbits are within the scope of the present invention. The term “lung condition” as used herein refers to a disease, event, or change in health status relating to the lung, including for example lung cancer and various non-cancerous conditions.

“Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value. Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus misclassified outcomes (false positives (FP) or false negatives (FN)), and may be stated as a sensitivity, specificity, positive predictive values (PPV) or negative predictive values (NPV), or as a likelihood, odds ratio, among other measures.

The term “biological sample” as used herein refers to any sample of biological origin potentially containing one or more biomarker proteins. Examples of biological samples include tissue, organs, or bodily fluids such as whole blood, plasma, serum, tissue, lavage or any other specimen used for detection of disease.

The term “subject” as used herein refers to a mammal, preferably a human.

The term “biomarker protein” as used herein refers to a polypeptide in a biological sample from a subject with a lung condition versus a biological sample from a control subject. A biomarker protein includes not only the polypeptide itself, but also minor variations thereof, including for example one or more amino acid substitutions or modifications such as glycosylation or phosphorylation.

The term “biomarker protein panel” as used herein refers to a plurality of biomarker proteins. In certain embodiments, the expression levels of the proteins in the panels can be correlated with the existence of a lung condition in a subject. In certain embodiments, biomarker protein panels comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90 or 100 proteins. In certain embodiments, the biomarker proteins panels comprise 2-5 proteins, 5-10 proteins, 10-20 proteins or more.

“Treating” or “treatment” as used herein with regard to a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.

Biomarker levels may change due to treatment of the disease. The changes in biomarker levels may be measured by the present invention. Changes in biomarker levels may be used to monitor the progression of disease or therapy.

“Altered”, “changed” or “significantly different” refer to a detectable change or difference from a reasonably comparable state, profile, measurement, or the like. One skilled in the art should be able to determine a reasonable measurable change. Such changes may be all or none. They may be incremental and need not be linear. They may be by orders of magnitude. A change may be an increase or decrease by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more, or any value in between 0% and 100%. Alternatively the change may be 1-fold, 1.5-fold 2-fold, 3-fold, 4-fold, 5-fold or more, or any values in between 1-fold and five-fold. The change may be statistically significant with a p value of 0.1, 0.05, 0.001, or 0.0001.

Using the methods of the current invention, a clinical assessment of a patient is first performed. If there exists is a higher likelihood for cancer, the clinician may rule in the disease which will require the pursuit of diagnostic testing options yielding data which increase and/or substantiate the likelihood of the diagnosis. “Rule in” of a disease requires a test with a high specificity.

“FN” is false negative, which for a disease state test means classifying a disease subject incorrectly as non-disease or normal.

“FP” is false positive, which for a disease state test means classifying a normal subject incorrectly as having disease.

The term “rule in” refers to a diagnostic test with high specificity that optionally coupled with a clinical assessment indicates a higher likelihood for cancer. If the clinical assessment is a lower likelihood for cancer, the clinician may adopt a stance to rule out the disease, which will require diagnostic tests which yield data that decrease the likelihood of the diagnosis. “Rule out” requires a test with a high sensitivity. Accordingly, the term “ruling in” as used herein is meant that the subject is selected to receive a treatment protocol.

The term “rule out” refers to a diagnostic test with high sensitivity that optionally coupled with a clinical assessment indicates a lower likelihood for cancer. Accordingly, the term “ruling out” as used herein is meant that the subject is selected not to receive a treatment protocol.

The term “sensitivity of a test” refers to the probability that a patient with the disease will have a positive test result. This is derived from the number of patients with the disease who have a positive test result (true positive) divided by the total number of patients with the disease, including those with true positive results and those patients with the disease who have a negative result, i.e. false negative.

The term “specificity of a test” refers to the probability that a patient without the disease will have a negative test result. This is derived from the number of patients without the disease who have a negative test result (true negative) divided by all patients without the disease, including those with a true negative result and those patients without the disease who have a positive test result, e.g. false positive. While the sensitivity, specificity, true or false positive rate, and true or false negative rate of a test provide an indication of a test's performance, e.g. relative to other tests, to make a clinical decision for an individual patient based on the test's result, the clinician requires performance parameters of the test with respect to a given population.

The term “positive predictive value” (PPV) refers to the probability that a positive result correctly identifies a patient who has the disease, which is the number of true positives divided by the sum of true positives and false positives.

The term “negative predictive value” or “NPV” is calculated by TN/(TN+FN) or the true negative fraction of all negative test results. It also is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested. The term NPV refers to the probability that a negative test correctly identifies a patient without the disease, which is the number of true negatives divided by the sum of true negatives and false negatives. A positive result from a test with a sufficient PPV can be used to rule in the disease for a patient, while a negative result from a test with a sufficient NPV can be used to rule out the disease, if the disease prevalence for the given population, of which the patient can be considered a part, is known.

The term “disease prevalence” refers to the number of all new and old cases of a disease or occurrences of an event during a particular period. Prevalence is expressed as a ratio in which the number of events is the numerator and the population at risk is the denominator.

The term disease incidence refers to a measure of the risk of developing some new condition within a specified period of time; the number of new cases during some time period, it is better expressed as a proportion or a rate with a denominator.

Lung cancer risk according to the “National Lung Screening Trial” is classified by age and smoking history. High risk—age ≧55 and ≧30 pack-years smoking history; Moderate risk—age ≧50 and ≧20 pack-years smoking history; Low risk—<age 50 or <20 pack-years smoking history.

The clinician must decide on using a diagnostic test based on its intrinsic performance parameters, including sensitivity and specificity, and on its extrinsic performance parameters, such as positive predictive value and negative predictive value, which depend upon the disease's prevalence in a given population.

Additional parameters which may influence clinical assessment of disease likelihood include the prior frequency and closeness of a patient to a known agent, e.g. exposure risk, that directly or indirectly is associated with disease causation, e.g. second hand smoke, radiation, etc., and also the radiographic appearance or characterization of the pulmonary nodule exclusive of size. A nodule's description may include solid, semi-solid or ground glass which characterizes it based on the spectrum of relative gray scale density employed by the CT scan technology.

“Mass spectrometry” refers to a method comprising employing an ionization source to generate gas phase ions from an analyte presented on a sample presenting surface of a probe and detecting the gas phase ions with a mass spectrometer.

In an embodiment of the invention, a panel of 5 proteins (ALDOA, FRIL, LG3BP, TSP1, and COIA1) and one protein-protein interaction term (FRIL and COIA1) effectively distinguish between samples derived from patients with benign and malignant nodules less than 2 cm diameter.

Bioinformatic and biostatistical analyses were used first to identify individual proteins with statistically significant differential expression, and then using these proteins to derive one or more combinations of proteins or panels of proteins, which collectively demonstrated superior discriminatory performance compared to any individual protein. Bioinformatic and biostatistical methods are used to derive coefficients (C) for each individual protein in the panel that reflects its relative expression level, i.e. increased or decreased, and its weight or importance with respect to the panel's net discriminatory ability, relative to the other proteins. The quantitative discriminatory ability of the panel can be expressed as a mathematical algorithm with a term for each of its constituent proteins being the product of its coefficient and the protein's plasma expression level (P) (as measured by LC-SRM-MS), e.g. C×P, with an algorithm consisting of n proteins described as: C1×P1+C2×P2+C3×P3+ . . . +Cn×Pn. An algorithm that discriminates between disease states with a predetermined level of statistical significance may be refers to a “disease classifier”. In addition to the classifier's constituent proteins with differential expression, it may also include proteins with minimal or no biologic variation to enable assessment of variability, or the lack thereof, within or between clinical specimens; these proteins may be termed typical native proteins and serve as internal controls for the other classifier proteins.

In certain embodiments, expression levels are measured by MS. MS analyzes the mass spectrum produced by an ion after its production by the vaporization of its parent protein and its separation from other ions based on its mass-to-charge ratio. The most common modes of acquiring MS data are 1) full scan acquisition resulting in the typical total ion current plot (TIC), 2) selected ion monitoring (SIM), and 3) selected reaction monitoring (SRM).

In certain embodiments of the methods provided herein, biomarker protein expression levels are measured by LC-SRM-MS. LC-SRM-MS is a highly selective method of tandem mass spectrometry which has the potential to effectively filter out all molecules and contaminants except the desired analyte(s). This is particularly beneficial if the analysis sample is a complex mixture which may comprise several isobaric species within a defined analytical window. LC-SRM-MS methods may utilize a triple quadrupole mass spectrometer which, as is known in the art, includes three quadrupole rod sets. A first stage of mass selection is performed in the first quadrupole rod set, and the selectively transmitted ions are fragmented in the second quadrupole rod set. The resultant transition (product) ions are conveyed to the third quadrupole rod set, which performs a second stage of mass selection. The product ions transmitted through the third quadrupole rod set are measured by a detector, which generates a signal representative of the numbers of selectively transmitted product ions. The RF and DC potentials applied to the first and third quadrupoles are tuned to select (respectively) precursor and product ions that have m/z values lying within narrow specified ranges. By specifying the appropriate transitions (m/z values of precursor and product ions), a peptide corresponding to a targeted protein may be measured with high degrees of sensitivity and selectivity. Signal-to-noise ratio is superior to conventional tandem mass spectrometry (MS/MS) experiments, which select one mass window in the first quadrupole and then measure all generated transitions in the ion detector. LC-SRM-MS.

In certain embodiments, an SRM-MS assay for use in diagnosing or monitoring lung cancer as disclosed herein may utilize one or more peptides and/or peptide transitions derived from the proteins ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN. In certain embodiments, the peptides and/or peptide transitions derived from 2 or more proteins “interact” mathematically. In certain embodiments, the peptides and/or peptide transitions derived from FRIL and COIA1 mathematically interact in the model for determining the probability score of lung cancer.

The expression level of a biomarker protein can be measured using any suitable method known in the art, including but not limited to mass spectrometry (MS), reverse transcriptase-polymerase chain reaction (RT-PCR), microarray, serial analysis of gene expression (SAGE), gene expression analysis by massively parallel signature sequencing (MPSS), immunoassays (e.g., ELISA), immunohistochemistry (IHC), transcriptomics, and proteomics.

To evaluate the diagnostic performance of a particular set of peptide transitions, a ROC curve is generated for each significant transition.

An “ROC curve” as used herein refers to a plot of the true positive rate (sensitivity) against the false positive rate (specificity) for a binary classifier system as its discrimination threshold is varied. A ROC curve can be represented equivalently by plotting the fraction of true positives out of the positives (TPR=true positive rate) versus the fraction of false positives out of the negatives (FPR=false positive rate). Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold.

AUC represents the area under the ROC curve. The AUC is an overall indication of the diagnostic accuracy of 1) a biomarker or a panel of biomarkers and 2) a ROC curve. AUC is determined by the “trapezoidal rule.” For a given curve, the data points are connected by straight line segments, perpendiculars are erected from the abscissa to each data point, and the sum of the areas of the triangles and trapezoids so constructed is computed. In certain embodiments of the methods provided herein, a biomarker protein has an AUC in the range of about 0.75 to 1.0. In certain of these embodiments, the AUC is in the range of about 0.8 to 0.8, 0.9 to 0.95, or 0.95 to 1.0.

The methods provided herein are minimally invasive and pose little or no risk of adverse effects. As such, they may be used to diagnose, monitor and provide clinical management of subjects who do not exhibit any symptoms of a lung condition and subjects classified as low risk for developing a lung condition. For example, the methods disclosed herein may be used to diagnose lung cancer in a subject who does not present with a PN and/or has not presented with a PN in the past, but who nonetheless deemed at risk of developing a PN and/or a lung condition. Similarly, the methods disclosed herein may be used as a strictly precautionary measure to diagnose healthy subjects who are classified as low risk for developing a lung condition.

The present invention provides a method of determining the likelihood that a lung condition in a subject is cancer by measuring an abundance of a panel of proteins in a sample obtained from the subject; calculating a probability of cancer score based on the protein measurements and ruling out cancer for the subject if the score is lower than a pre-determined score, when cancer is ruled out the subject does not receive a treatment protocol. Treatment protocols include for example pulmonary function test (PFT), pulmonary imaging, a biopsy, a surgery, a chemotherapy, a radiotherapy, or any combination thereof. In some embodiments, the imaging is an x-ray, a chest computed tomography (CT) scan, or a positron emission tomography (PET) scan.

The present invention further provides a method of ruling in the likelihood of cancer for a subject by measuring an abundance of panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and ruling in the likelihood of cancer for the subject if the score is higher than a pre-determined score.

In another aspect the invention further provides a method of determining the likelihood of the presence of a lung condition in a subject by measuring an abundance of panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and concluding the presence of this lung condition if the score is equal or greater than a pre-determined score. The lung condition is lung cancer such as for example, non-small cell lung cancer (NSCLC). The subject is at risk of developing lung cancer.

The panel includes 5 proteins ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN. Nucleic acid and amino acid sequences for these can be found in Table 6 and Table 7, respectively. Preferably, FRIL_HUMAN and COIA1_HUMAN mathematically interact in the model for determining the probability score.

In merely illustrative embodiments, the methods described herein include steps of (a) measuring the abundance (intensity) of one representative peptide transition derived from each of the proteins comprising ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN in a sample obtained from a subject; (b) determining the coefficient for each representative peptide transition; (c) calculating a sum of the products of each logarithmically transformed (and optionally normalized) intensity of each transition and its corresponding coefficient; (d) calculating a mathematical interaction between FRIL and COIA1 by multiplying their logarithmically transformed (and optionally normalized) intensity of their representative peptide transitions; and (e) calculating a probability of cancer score based on the sum calculated in step (c) and the mathematical interaction calculated in step (d).

In some embodiments, the representative peptide transitions for proteins ALDOA_HUMAN, COIA1_HUMAN, TSP1_HUMAN, FRIL_HUMAN, and LG3BP_HUMAN are ALQASALK (SEQ ID NO: 25) (401.25, 617.4), AVGLAGTFR (SEQ ID NO: 26) (446.26, 721.4), GFLLLASLR (SEQ ID NO: 27) (495.31, 559.4), LGGPEAGLGEYLFER (SEQ ID NO: 28) (804.4, 1083.6), and VEIFYR (SEQ ID NO: 29) (413.73, 598.3), respectively.

In some embodiments, the measuring step of any method described herein is performed by detecting transitions comprising ALQASALK (SEQ ID NO: 25) (401.25, 617.4), AVGLAGTFR (SEQ ID NO: 26) (446.26, 721.4), GFLLLASLR (SEQ ID NO: 27) (495.31, 559.4), LGGPEAGLGEYLFER (SEQ ID NO: 28) (804.4, 1083.6), and VEIFYR (SEQ ID NO: 29) (413.73, 598.3).

The subject has or is suspected of having a pulmonary nodule. The pulmonary nodule has a diameter of less than or equal to 3.0 cm. In one embodiment, the pulmonary nodule has a diameter of about 0.8 cm to 2.0 cm. The subject may have stage IA lung cancer (i.e., the tumor is smaller than 3 cm).

The probability score is calculated from a logistic regression model applied to the protein measurements. For example, the score is determined as P_(s)=1/[1+exp(−α−Σ_(i=1) ⁵β_(i)*{hacek over (I)}_(i,s)−γ*{hacek over (I)}_(COIA1)*{hacek over (I)}_(FRIL))], where {hacek over (I)}_(i,s) is logarithmically transformed and normalized intensity of transition i in said sample (s), β_(i) is the corresponding logistic regression coefficient, α is a panel-specific constant, and γ is a coefficient for the interaction term. The score determined has a negative predictive value (NPV) of at least about 85%, at least 90% or higher (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher).

In various embodiments, the method of the present invention further comprises normalizing the protein measurements. For example, the protein measurements are normalized by one or more proteins selected from PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN and PTPRJ_HUMAN. Nucleic acid and amino acid sequences for these can be found in Table 8 and Table 9, respectively.

The biological sample includes such as for example tissue, blood, plasma, serum, whole blood, urine, saliva, genital secretion, cerebrospinal fluid, sweat and excreta.

In some embodiments, the determining the likelihood of cancer is determined by the sensitivity, specificity, negative predictive value or positive predictive value associated with the score.

The measuring step is performed by selected reaction monitoring mass spectrometry, using a compound that specifically binds the protein being detected or a peptide transition. In one embodiment, the compound that specifically binds to the protein being measured is an antibody or an aptamer.

In specific embodiments, the diagnostic methods disclosed herein are used to rule out a treatment protocol for a subject, measuring the abundance of a panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and protein-protein interaction and ruling out the treatment protocol for the subject if the score determined in the sample is lower than a pre-determined score. In some embodiments the panel contains ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN; and FRIL_HUMAN and COIA1_HUMAN interact in the model for determining the score.

In specific embodiments, the diagnostic methods disclosed herein are used to rule in a treatment protocol for a subject by measuring the abundance of a panel of proteins in a sample obtained from the subject, calculating a probability of cancer score based on the protein measurements and protein-protein interaction and ruling in the treatment protocol for the subject if the score determined in the sample is greater than a pre-determined score. In some embodiments the panel contains ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, and COIA1_HUMAN; and FRIL_HUMAN and COIA1_HUMAN interact in the model for determining the score.

In certain embodiments, the diagnostic methods disclosed herein can be used in combination with other clinical assessment methods, including for example various radiographic and/or invasive methods. Similarly, in certain embodiments, the diagnostic methods disclosed herein can be used to identify candidates for other clinical assessment methods, or to assess the likelihood that a subject will benefit from other clinical assessment methods.

The high abundance of certain proteins in a biological sample such as plasma or serum can hinder the ability to assay a protein of interest, particularly where the protein of interest is expressed at relatively low concentrations. Several methods are available to circumvent this issue, including enrichment, separation, and depletion. Enrichment uses an affinity agent to extract proteins from the sample by class, e.g., removal of glycosylated proteins by glycocapture. Separation uses methods such as gel electrophoresis or isoelectric focusing to divide the sample into multiple fractions that largely do not overlap in protein content. Depletion typically uses affinity columns to remove the most abundant proteins in blood, such as albumin, by utilizing advanced technologies such as IgY14/Supermix (SigmaSt. Louis, Mo.) that enable the removal of the majority of the most abundant proteins.

In certain embodiments of the methods provided herein, a biological sample may be subjected to enrichment, separation, and/or depletion prior to assaying biomarker or putative biomarker protein expression levels. In certain of these embodiments, blood proteins may be initially processed by a glycocapture method, which enriches for glycosylated proteins, allowing quantification assays to detect proteins in the high pg/ml to low ng/ml concentration range. Exemplary methods of glycocapture are well known in the art (see, e.g., U.S. Pat. No. 7,183,188; U.S. Patent Appl. Publ. No. 2007/0099251; U.S. Patent Appl. Publ. No. 2007/0202539; U.S. Patent Appl. Publ. No. 2007/0269895; and U.S. Patent Appl. Publ. No. 2010/0279382). In other embodiments, blood proteins may be initially processed by a protein depletion method, which allows for detection of commonly obscured biomarkers in samples by removing abundant proteins. In one such embodiment, the protein depletion method is a Supermix (Sigma) depletion method.

In certain embodiments, a biomarker protein panel comprises two to 100 biomarker proteins. In certain of these embodiments, the panel comprises 2 to 5, 6 to 10, 11 to 15, 16 to 20, 21-25, 5 to 25, 26 to 30, 31 to 40, 41 to 50, 25 to 50, 51 to 75, 76 to 100, biomarker proteins. In certain embodiments, a biomarker protein panel comprises one or more subpanels of biomarker proteins that each comprises at least two biomarker proteins. For example, biomarker protein panel may comprise a first subpanel made up of biomarker proteins that are overexpressed in a particular lung condition and a second subpanel made up of biomarker proteins that are under-expressed in a particular lung condition.

In certain embodiments of the methods, compositions, and kits provided herein, a biomarker protein may be a protein that exhibits differential expression in conjunction with lung cancer.

In other embodiments, the diagnosis methods disclosed herein may be used to distinguish between two different lung conditions. For example, the methods may be used to classify a lung condition as malignant lung cancer versus benign lung cancer, NSCLC versus SCLC, or lung cancer versus non-cancer condition (e.g., inflammatory condition).

In certain embodiments, kits are provided for diagnosing a lung condition in a subject. These kits are used to detect expression levels of one or more biomarker proteins. Optionally, a kit may comprise instructions for use in the form of a label or a separate insert. The kits can contain reagents that specifically bind to proteins in the panels described, herein. These reagents can include antibodies. The kits can also contain reagents that specifically bind to mRNA expressing proteins in the panels described, herein. These reagents can include nucleotide probes. The kits can also include reagents for the detection of reagents that specifically bind to the proteins in the panels described herein. These reagents can include fluorophores.

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

EXAMPLES Example 1 Identification of a Robust Classifier that Distinguishes Malignant and Benign Lung Nodule

Plasma samples of patients originated from three sites (UPenn, Laval and NYU) were divided into five experimental batches. Within each batch, four aliquots of a pooled human plasma standard (HPS) sample were processed. Plasma samples were immuno-depleted, denatured, reduced, trypsin-digested, and analyzed by LC-MRM-MS at Integrated Diagnostics using protocols developed in previous studies.

The 100 clinical samples were all from patients with lung nodules of 8-20 mm in size and age >40 years. Cancer and benign samples were matched on gender, age (+/−10 years) and nodule size (+/−8 mm). There were some bias between cancer and benign samples on smoking history and on smoking pack-years.

TABLE 1 Sources of samples and their assignment to five batches. Batch Center Benign Cancer Total S1 UPenn 10 10 20 S2 UPenn 10 10 20 S3 Laval 10 10 20 S4 NYU 10 10 20 S5 NYU 10 10 20 Total 3 Sites 50 50 100

Detailed procedures for sample preparation and data processing, including normalization of the raw data can be found in PCT/US2012/071387 (WO13/096845), the contents of which are incorporated herein by their entireties.

Among all the possible panels formed by the 13 proteins identified in WO13/096845, there were 28 panels with a cross-validated performance with partial AUC at specificity=0.9 greater than two-fold the number expected by random chance (0.1^2/2). These models were retained and using 100,000 cross-validation models to get a more accurate measure of their logistic regression coefficients and to determine the coefficient of variability for the model coefficients. Measure the CVs of each protein coefficient and report the NPV, SPC performance, on median panel was performed at a prevalence of 20%.

TABLE 2 Robust 28 panels Proteins max_cv max_cv_protein ALPHA_CV NPV specificity threshold xv_pAUC_factor ALDOA, TSP1, PRDX1, LG3BP 0.54 ALDOA 0.73 0.90 0.68 0.50 3.33 ALDOA, TSP1, LG3BP 0.58 TSP1 0.73 0.90 0.55 0.49 4.47 ALDOA, COIA1, TSP1, LG3BP 0.73 COIA1 0.62 0.90 0.55 0.49 4.17 ALDOA, COIA1, FRIL, LG3BP 0.62 COIA1 0.38 0.90 0.51 0.48 3.89 COIA1, LG3BP 0.78 COIA1 0.57 0.90 0.51 0.49 3.75 LG3BP 0.23 LG3BP 0.32 0.90 0.49 0.48 4.05 ALDOA, LG3BP 0.44 ALDOA 0.38 0.91 0.47 0.47 5.45 ALDOA, LRP1, LG3BP 0.54 LRP1 0.66 0.91 0.47 0.46 4.26 ALDOA, COIA1, PRDX1, LG3BP 0.73 ALDOA 0.75 0.90 0.45 0.45 3.82 COIA1, PRDX1, LG3BP 0.70 COIA1 0.89 0.90 0.43 0.45 3.35 ALDOA, COIA1, LG3BP 0.65 COIA1 0.52 0.90 0.38 0.45 5.26 ISLR, ALDOA, COIA1, TSP1, 6.85 COIA1 0.96 0.90 0.72 0.49 2.10 FRIL, PRDX1, LRP1, LG3BP PRDX1, LG3BP 0.37 PRDX1 1.50 0.90 0.55 0.49 3.34 ALDOA, PRDX1, LG3BP 0.82 ALDOA 2.61 0.90 0.53 0.47 3.74 ISLR, ALDOA, TSP1, PRDX1, LG3BP 1.50 ISLR 2.00 0.90 0.53 0.48 3.31 ISLR, ALDOA, COIA1, TSP1, PRDX1, LG3BP 42.98 ISLR 4.48 0.90 0.53 0.48 2.90 ISLR, ALDOA, TSP1, LG3BP 1.13 ISLR 1.04 0.90 0.51 0.48 4.08 ISLR, ALDOA, COIA1, TSP1, LG3BP 4.33 ISLR 1.50 0.90 0.51 0.48 3.76 ISLR, ALDOA, PRDX1, LG3BP 1.17 ISLR 1.24 0.90 0.51 0.47 3.74 ISLR, LG3BP 1.18 ISLR 1.01 0.91 0.47 0.47 3.57 ISLR, COIA1, LG3BP 4.46 ISLR 1.43 0.91 0.47 0.48 3.30 ISLR, PRDX1, LG3BP 1.32 ISLR 1.46 0.91 0.47 0.46 3.28 ISLR, ALDOA, LG3BP 1.01 ISLR 0.89 0.90 0.45 0.46 4.91 ALDOA, COIA1, LRP1, LG3BP 0.83 COIA1 3.18 0.90 0.45 0.46 4.01 ISLR, ALDOA, COIA1, PRDX1, LG3BP 8.97 ISLR 2.14 0.90 0.45 0.45 3.58 ISLR, COIA1, PRDX1, LG3BP 20.54 ISLR 2.86 0.90 0.43 0.45 3.12 ISLR, ALDOA, COIA1, LG3BP 3.63 ISLR 1.27 0.90 0.38 0.44 4.71 ISLR, ALDOA, LRP1, LG3BP 0.95 ISLR 2.97 0.90 0.38 0.44 3.97

All possible panels of proteins ALDOA, COIA1, FRIL, LG3BP, LRP1, PRDX1, TSP1, TETN, and BGH3 are next generated. A set of 27 panels were selected to be carried forward by the following criteria:

-   -   Median Specificity>=0.5     -   Max Coefficient CV<=1.5     -   Maximum ALPHA CV<=1.5     -   Cross-validated pAUC at specificity=0.9 greater than one fold         random.     -   A minimum of four proteins per panel.

The top 6 panels were carried forward.

TABLE 3 Top 6 panels Median Panel Proteins Size Specificity Rank xv_Specificity ID_341 ALDOA, TSP1, FRIL, PRDX1, 5 0.62 3 0.32 LG3BP ID_85 TSP1, FRIL, PRDX1, LG3BP 4 0.55 5 0.31 ID_340 ALDOA, TSP1, FRIL, PRDX1 4 0.66 1 0.29 ID_449 ALDOA, COIA1, TSP1, LG3BP 4 0.51 6 0.27 ID_465 ALDOA, COIA1, TSP1, FRIL, 5 0.60 4 0.24 LG3BP ID_469 ALDOA, COIA1, TSP1, FRIL, 6 0.64 2 0.23 PRDX1, LG3BP

Representative NPV/Specificity plot for ID_(—)465 and ID_(—)341 panels can be found in FIGS. 2 and 3, respectively.

All possible interaction pairs were added to panel 465. The cross validated performance (Specificity at NPV=0.90) and partial AUC was measured. The below table displays the performance:

Cross Validated Performance and Partial AUC for Panel 465.

xv_(—) ID_465 ID465 Max_cv_(—) ALPHA_(—) Median Median pAUC_(—) xv_(—) xv_(—) xv- xv_(—) Name Max_cv protein CV NPV specificity threshold xv NPV Spec Threshold spec pAUC ID_4 0.981 Interaction 0.429 0.901 0.617 0.483 1.751 0.9 0.182 0.346 0 0 65 term 00 ALQA SALK _401 .25_(—) 617. 40_(—) time s_AV GLAG TFR_(—) 446. 26_7 21.4 0 ID_4 0.955 GFLLLASLR_4 0.381 0.904 0.638 0.481 1.571 0.9 0.201 0.355 0 0 65 95.31_559.4 00 ALQA 0 SALK _401 .25_(—) 617. 40_(—) time s_GF LLLA SLR_(—) 495. 31_5 59.4 0 ID_4 0.735 LGGPEAGLGEY 0.529 0.901 0.681 0.501 1.944 0.9 0.240 0.375 0 0 65 LFER_804.40 00 ALQA _1083.60 SALK _401 .25_(—) 617. 40_(—) time s_LG GPEA GLGE YLFE R_80 4.40 _108 3.60 ID_4 0.953 Interaction 0.397 0.901 0.617 0.495 2.209 0.9 0.241 0.376 0 1 65 term 00 ALQA SALK _401 .25_(—) 617. 40_(—) time s_VE IFYR _413 .73_(—) 598. 30 ID_4 0.891 Interaction 0.475 0.901 0.511 0.455 1.734 0.9 0.188 0.336 0 0 65 term 00 AVGL AGTF R_44 6.26 _721 .40_(—) time s_GF LLLA SLR_(—) 495. 31_5 59.4 0 ID_4 0.466 LGGPEAGLGEY 0.619 0.902 0.660 0.496 2.402 0.9 0.396 0.422 1 1 65 LFER_804.40 00 AVGL _1083.60 AGTF R_44 6.26 _721 .40_(—) time s_LG GPEA GLGE YLFE R_80 4.40 _108 3.60 ID_4 4.349 VEIFYR_413. 0.510 0.905 0.574 0.481 1.643 0.9 0.216 0.360 0 0 65 73_598.30 00 AVGL AGTF R_44 6.26 _721 .40_(—) time s_VE IFYR _413 .73_(—) 598. 30 ID_4 556.51 Interaction 0.420 0.901 0.617 0.485 1.217 0.9 0.165 0.337 0 0 65 0 term 00 GFLL LASL R_49 5.31 _559 .40_(—) time s_LG GPEA GLGE YLFE R_80 4.40 _108 3.60 ID_4 0.806 AVGLAGTFR_4 0.392 0.903 0.702 0.509 1.955 0.9 0.222 0.370 0 0 65 46.26_721.4 00 GFLL 0 LASL R_49 5.31 _559 .40_(—) time s_VE IFYR _413 .73_(—) 598. 30 ID_4 0.743 AVGLAGTFR_4 0.387 0.902 0.660 0.496 1.947 0.9 0.283 0.392 1 0 65 46.26_721.4 00 LGGP 0 EAGL GEYL FER_(—) 804. 40_1 083. 60_t imes _VEI FYR_(—) 413. 73_5 98.3 0 ID_4 0.700 AVGLAGTFR_4 0.404 0.903 0.596 0.482 1.974 0.9 0.246 0.381 65 46.26_721.4 00 0

The panel including the interaction term from COIA1 and FRIL performed much better than the panel without interaction terms in both cross validated specificity at NPV=0.9 and cross validated partial AUC.

TABLE 4 C4 Classifier SEQ ID Precursor Protein Compound Name NO: Ion Product Ion Coefficient ALDOA_HUMAN ALQASALK 25 401.25 617.4 −0.47459794 (Beta) COIA1_HUMAN AVGLAGTFR 26 446.26 721.4 −2.468073083 (Beta) TSP1_HUMAN GFLLLASLR 27 495.31 559.4  0.33223188 (Beta) FRIL_HUMAN LGGPEAGLGEYLFER 28 804.4 1083.6 −0.864887827 LG3BP_HUMAN VEIFYR 29 413.73 598.3 −0.903170248 COIA1 × FRIL Interaction −1.227671396 ALPHA Constant −1.621210001

TABLE 5 Performance of C4 Classifier Threshold NPV Specificity 0.48 0.85 0.55 0.37 0.90 0.28 0.27 0.95* 0.13

TABLE 6 Nucleotide sequences of proteins in high performing panels. Seq. Gene Name Nucleotide Sequence ID. ALDOA_HUMAN ATGCCCTACCAATATCCAGCACTGACCCCGGAGCAGAAGAAGGAGCTGTCT 1 GAGATCGCTCACCGCATCGTGGCACCTGGCAAGGGCATCCTGGCTGCAGAT GAGTCCACTGGGAGCATTGCCAAGCGGCTGCAGTCCATTGGCACCGAGAAC ACCGAGGAGAACCGGCGCTTCTACCGCCAGCTGCTGCTGACAGCTGACGAC CGCGTGAACCCCTGCATTGGGGGTGTCATCCTCTTCCATGAGACACTCTAC CAGAAGGCGGATGATGGGCGTCCCTTCCCCCAAGTTATCAAATCCAAGGGC GGTGTTGTGGGCATCAAGGTAGACAAGGGCGTGGTCCCCCTGGCAGGGACA AATGGCGAGACTACCACCCAAGGGTTGGATGGGCTGTCTGAGCGCTGTGCC CAGTACAAGAAGGACGGAGCTGACTTCGCCAAGTGGCGTTGTGTGCTGAAG ATTGGGGAACACACCCCCTCAGCCCTCGCCATCATGGAAAATGCCAATGTT CTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCCATCGTG GAGCCTGAGATCCTCCCTGATGGGGACCATGACTTGAAGCGCTGCCAGTAT GTGACCGAGAAGGTGCTGGCTGCTGTCTACAAGGCTCTGAGTGACCACCAC ATCTACCTGGAAGGCACCTTGCTGAAGCCCAACATGGTCACCCCAGGCCAT GCTTGCACTCAGAAGTTTTCTCATGAGGAGATTGCCATGGCGACCGTCACA GCGCTGCGCCGCACAGTGCCCCCCGCTGTCACTGGGATCACCTTCCTGTCT GGAGGCCAGAGTGAGGAGGAGGCGTCCATCAACCTCAATGCCATTAACAAG TGCCCCCTGCTGAAGCCCTGGGCCCTGACCTTCTCCTACGGCCGAGCCCTG CAGGCCTCTGCCCTGAAGGCCTGGGGCGGGAAGAAGGAGAACCTGAAGGCT GCGCAGGAGGAGTATGTCAAGCGAGCCCTGGCCAACAGCCTTGCCTGTCAA GGAAAGTACACTCCGAGCGGTCAGGCTGGGGCTGCTGCCAGCGAGTCCCTC TTCGTCTCTAACCACGCCTATTAA ALDOA_HUMAN ATGGCAAGGCGCAAGCCAGAAGGGTCCAGCTTCAACATGACCCACCTGTCC 2 (isoform ATGGCTATGGCCTTTTCCTTTCCCCCAGTTGCCAGTGGGCAACTCCACCCT 2) CAGCTGGGCAACACCCAGCACCAGACAGAGTTAGGAAAGGAACTTGCTACT ACCAGCACCATGCCCTACCAATATCCAGCACTGACCCCGGAGCAGAAGAAG GAGCTGTCTGACATCGCTCACCGCATCGTGGCACCTGGCAAGGGCATCCTG GCTGCAGATGAGTCCACTGGGAGCATTGCCAAGCGGCTGCAGTCCATTGGC ACCGAGAACACCGAGGAGAACCGGCGCTTCTACCGCCAGCTGCTGCTGACA GCTGACGACCGCGTGAACCCCTGCATTGGGGGTGTCATCCTCTTCCATGAG ACACTCTACCAGAAGGCGGATGATGGGCGTCCCTTCCCCCAAGTTATCAAA TCCAAGGGCGGTGTTGTGGGCATCAAGGTAGACAAGGGCGTGGTCCCCCTG GCAGGGACAAATGGCGAGACTACCACCCAAGGGTTGGATGGGCTGTCTGAG CGCTGTGCCCAGTACAAGAAGGACGGAGCTGACTTCGCCAAGTGGCGTTGT GTGCTGAAGATTGGGGAACACACCCCCTCAGCCCTCGCCATCATGGAAAAT GCCAATGTTCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTG CCCATCGTGGAGCCTGAGATCCTCCCTGATGGGGACCATGACTTGAAGCGC TGCCAGTATGTGACCGAGAAGGTGCTGGCTGCTGTCTACAAGGCTCTGAGT GACCACCACATCTACCTGGAAGGCACCTTGCTGAAGCCCAACATGGTCACC CCAGGCCATGCTTGCACTCAGAAGTTTTCTCATGAGGAGATTGCCATGGCG ACCGTCACAGCGCTGCGCCGCACAGTGCCCCCCGCTGTCACTGGGATCACC TTCCTGTCTGGAGGCCAGAGTGAGGAGGAGGCGTCCATCAACCTCAATGCC ATTAACAAGTGCCCCCTGCTGAAGCCCTGGGCCCTGACCTTCTCCTACGGC CGAGCCCTGCAGGCCTCTGCCCTGAAGGCCTGGGGCGGGAAGAAGGAGAAC CTGAAGGCTGCGCAGGAGGAGTATGTCAAGCGAGCCCTGGCCAACAGCCTT GCCTGTCAAGGAAAGTACACTCCGAGCGGTCAGGCTGGGGCTGCTGCCAGC GAGTCCCTCTTCGTCTCTAACCACGCCTATTAA FRIL_HUMAN ATGAGCTCCCAGATTCGTCAGAATTATTCCACCGACGTGGAGGCAGCCGTC 3 AACAGCCTGGTCAATTTGTACCTGCAGGCCTCCTACACCTACCTCTCTCTG GGCTTCTATTTCGACCGCGATGATGTGGCTCTGGAAGGCGTGAGCCACTTC TTCCGCGAATTGGCCGAGGAGAAGCGCGAGGGCTACGAGCGTCTCCTGAAG ATGCAAAACCAGCGTGGCGGCCGCGCTCTCTTCCAGGACATCAAGAAGCCA GCTGAAGATGAGTGGGGTAAAACCCCAGACGCCATGAAAGCTGCCATGGCC CTGGAGAAAAAGCTGAACCAGGCCCTTTTGGATCTTCATGCCCTGGGTTCT GCCCGCACGGACCCCCATCTCTGTGACTTCCTGGAGACTCACTTCCTAGAT GAGGAAGTGAAGCTTATCAAGAAGATGGGTGACCACCTGACCAACCTCCAC AGGCTGGGTGGCCCGGAGGCTGGGCTGGGCGAGTATCTCTTCGAAAGGCTC ACTCTCAAGCACGACTAA LG3BP_HUMAN ATGACCCCTCCGAGGCTCTTCTGGGTGTGGCTGCTGGTTGCAGGAACCCAA 4 GGCGTGAACGATGGTGACATGCGGCTGGCCGATGGGGGCGCCACCAACCAG GGCCGCGTGGAGATCTTCTACAGAGGCCAGTGGGGCACTGTGTGTGACAAC CTGTGGGACCTGACTGATGCCAGCGTCGTCTGCCGGGCCCTGGGCTTCGAG AACGCCACCCAGGCTCTGGGCAGAGCTGCCTTCGGGCAAGGATCAGGCCCC ATCATGCTGGATGAGGTCCAGTGCACGGGAACCGAGGCCTCACTGGCCGAC TGCAAGTCCCTGGGCTGGCTGAAGAGCAACTGCAGGCACGAGAGAGACGCT GGTGTGGTCTGCACCAATGAAACCAGGAGCACCCACACCCTGGACCTCTCC AGGGAGCTCTCGGAGGCCCTTGGCCAGATCTTTGACAGCCAGCGGGGCTGC GACCTGTCCATCAGCGTGAATGTGCAGGGCGAGGACGCCCTGGGCTTCTGT GGCCACACGGTCATCCTGACTGCCAACCTGGAGGCCCAGGCCCTGTGGAAG GAGCCGGGCAGCAATGTCACCATGAGTGTGGATGCTGAGTGTGTGCCCATG GTCAGGGACCTTCTCAGGTACTTCTACTCCCGAAGGATTGACATCACCCTG TCGTCAGTCAAGTGCTTCCACAAGCTGGCCTCTGCCTATGGGGCCAGGCAG CTGCAGGGCTACTGCGCAAGCCTCTTTGCCATCCTCCTCCCCCAGGACCCC TCGTTCCAGATGCCCCTGGACCTGTATGCCTATGCAGTGGCCACAGGGGAC GCCCTGCTGGAGAAGCTCTGCCTACAGTTCCTGGCCTGGAACTTCGAGGCC TTGACGCAGGCCGAGGCCTGGCCCAGTGTCCCCACAGACCTGCTCCAACTG CTGCTGCCCAGGAGCGACCTGGCGGTGCCCAGCGAGCTGGCCCTACTGAAG GCCGTGGACACCTGGAGCTGGGGGGAGCGTGCCTCCCATGAGGAGGTGGAG GGCTTGGTGGAGAAGATCCGCTTCCCCATGATGCTCCCTGAGGAGCTCTTT GAGCTGCAGTTCAACCTGTCCCTGTACTGGAGCCACGAGGCCCTGTTCCAG AAGAAGACTCTGCAGGCCCTGGAATTCCACACTGTGCCCTTCCAGTTGCTG GCCCGGTACAAAGGCCTGAACCTCACCGAGGATACCTACAAGCCCCGGATT TACACCTCGCCCACCTGGAGTGCCTTTGTGACAGACAGTTCCTGGAGTGCA CGGAAGTCACAACTGGTCTATCAGTCCAGACGGGGGCCTTTGGTCAAATAT TCTTCTGATTACTTCCAAGCCCCCTCTGACTACAGATACTACCCCTACCAG TCCTTCCAGACTCCACAACACCCCAGCTTCCTCTTCCAGGACAAGAGGGTG TCCTGGTCCCTGGTCTACCTCCCCACCATCCAGAGCTGCTGGAACTACGGC TTCTCCTGCTCCTCGGACGAGCTCCCTGTCCTGGGCCTCACCAAGTCTGGC GGCTCAGATCGCACCATTGCCTACGAAAACAAAGCCCTGATGCTCTGCGAA GGGCTCTTCGTGGCAGACGTCACCGATTTCGAGGGCTGGAAGGCTGCGATT CCCAGTGCCCTGGACACCAACAGCTCGAAGAGCACCTCCTCCTTCCCCTGC CCGGCAGGGCACTTCAACGGCTTCCGCACGGTCATCCGCCCCTTCTACCTG ACCAACTCCTCAGGTGTGGACTAG TSP1_HUMAN ATGGGGCTGGCCTGGGGACTAGGCGTCCTGTTCCTGATGCATGTGTGTGGC 5 ACCAACCGCATTCCAGAGTCTGGCGGAGACAACAGCGTGTTTGACATCTTT GAACTCACCGGGGCCGCCCGCAAGGGGTCTGGGCGCCGACTGGTGAAGGGC CCCGACCCTTCCAGCCCAGCTTTCCGCATCGAGGATGCCAACCTGATCCCC CCTGTGCCTGATGACAAGTTCCAAGACCTGGTGGATGCTGTGCGGGCAGAA AAGGGTTTCCTCCTTCTGGCATCCCTGAGGCAGATGAAGAAGACCCGGGGC ACGCTGCTGGCCCTGGAGCGGAAAGACCACTCTGGCCAGGTCTTCAGCGTG GTGTCCAATGGCAAGGCGGGCACCCTGGACCTCAGCCTGACCGTCCAAGGA AAGCAGCACGTGGTGTCTGTGGAAGAAGCTCTCCTGGCAACCGGCCAGTGG AAGAGCATCACCCTGTTTGTGCAGGAAGACAGGGCCCAGCTGTACATCGAC TGTGAAAAGATGGAGAATGCTGAGTTGGACGTCCCCATCCAAAGCGTCTTC ACCAGAGACCTGGCCAGCATCGCCAGACTCCGCATCGCAAAGGGGGGCGTC AATGACAATTTCCAGGGGGTGCTGCAGAATGTGAGGTTTGTCTTTGGAACC ACACCAGAAGACATCCTCAGGAACAAAGGCTGCTCCAGCTCTACCAGTGTC CTCCTCACCCTTGACAACAACGTGGTGAATGGTTCCAGCCCTGCCATCCGC ACTAACTACATTGGCCACAAGACAAAGGACTTGCAAGCCATCTGCGGCATC TCCTGTGATGAGCTGTCCAGCATGGTCCTGGAACTCAGGGGCCTGCGCACC ATTGTGACCACGCTGCAGGACAGCATCCGCAAAGTGACTGAAGAGAACAAA GAGTTGGCCAATGAGCTGAGGCGGCCTCCCCTATGCTATCACAACGGAGTT CAGTACAGAAATAACGAGGAATGGACTGTTGATAGCTGCACTGAGTGTCAC TGTCAGAACTCAGTTACCATCTGCAAAAAGGTGTCCTGCCCCATCATGCCC TGCTCCAATGCCACAGTTCCTGATGGAGAATGCTGTCCTCGCTGTTGGCCC AGCGACTCTGCGGACGATGGCTGGTCTCCATGGTCCGAGTGGACCTCCTGT TCTACGAGCTGTGGCAATGGAATTCAGCAGCGCGGCCGCTCCTGCGATAGC CTCAACAACCGATGTGAGGGCTCCTCGGTCCAGACACGGACCTGCCACATT CAGGAGTGTGACAAGAGATTTAAACAGGATGGTGGCTGGAGCCACTGGTCC CCGTGGTCATCTTGTTCTGTGACATGTGGTGATGGTGTGATCACAAGGATC CGGCTCTGCAACTCTCCCAGCCCCCAGATGAACGGGAAACCCTGTGAAGGC GAAGCGCGGGAGACCAAAGCCTGCAAGAAAGACGCCTGCCCCATCAATGGA GGCTGGGGTCCTTGGTCACCATGGGACATCTGTTCTGTCACCTGTGGAGGA GGGGTACAGAAACGTAGTCGTCTCTGCAACAACCCCACACCCCAGTTTGGA GGCAAGGACTGCGTTGGTGATGTAACAGAAAACCAGATCTGCAACAAGCAG GACTGTCCAATTGATGGATGCCTGTCCAATCCCTGCTTTGCCGGCGTGAAG TGTACTAGCTACCCTGATGGCAGCTGGAAATGTGGTGCTTGTCCCCCTGGT TACAGTGGAAATGGCATCCAGTGCACAGATGTTGATGAGTGCAAAGAAGTG CCTGATGCCTGCTTCAACCACAATGGAGAGCACCGGTGTGAGAACACGGAC CCCGGCTACAACTGCCTGCCCTGCCCCCCACGCTTCACCGGCTCACAGCCC TTCGGCCAGGGTGTCGAACATGCCACGGCCAACAAACAGGTGTGCAAGCCC CGTAACCCCTGCACGGATGGGACCCACGACTGCAACAAGAACGCCAAGTGC AACTACCTGGGCCACTATAGCGACCCCATGTACCGCTGCGAGTGCAAGCCT GGCTACGCTGGCAATGGCATCATCTGCGGGGAGGACACAGACCTGGATGGC TGGCCCAATGAGAACCTGGTGTGCGTGGCCAATGCGACTTACCACTGCAAA AAGGATAATTGCCCCAACCTTCCCAACTCAGGGCAGGAAGACTATGACAAG GATGGAATTGGTGATGCCTGTGATGATGACGATGACAATGATAAAATTCCA GATGACAGGGACAACTGTCCATTCCATTACAACCCAGCTCAGTATGACTAT GACAGAGATGATGTGGGAGACCGCTGTGACAACTGTCCCTACAACCACAAC CCAGATCAGGCAGACACAGACAACAATGGGGAAGGAGACGCCTGTGCTGCA GACATTGATGGAGACGGTATCCTCAATGAACGGGACAACTGCCAGTACGTC TACAATGTGGACCAGAGAGACACTGATATGGATGGGGTTGGAGATCAGTGT GACAATTGCCCCTTGGAACACAATCCGGATCAGCTGGACTCTGACTCAGAC CGCATTGGAGATACCTGTGACAACAATCAGGATATTGATGAAGATGGCCAC CAGAACAATCTGGACAACTGTCCCTATGTGCCCAATGCCAACCAGGCTGAC CATGACAAAGATGGCAAGGGAGATGCCTGTGACCACGATGATGACAACGAT GGCATTCCTGATGACAAGGACAACTGCAGACTCGTGCCCAATCCCGACCAG AAGGACTCTGACGGCGATGGTCGAGGTGATGCCTGCAAAGATGATTTTGAC CATGACAGTGTGCCAGACATCGATGACATCTGTCCTGAGAATGTTGACATC AGTGAGACCGATTTCCGCCGATTCCAGATGATTCCTCTGGACCCCAAAGGG ACATCCCAAAATGACCCTAACTGGGTTGTACGCCATCAGGGTAAAGAACTC GTCCAGACTGTCAACTGTGATCCTGGACTCGCTGTAGGTTATGATGAGTTT AATGCTGTGGACTTCAGTGGCACCTTCTTCATCAACACCGAAAGGGACGAT GACTATGCTGGATTTGTCTTTGGCTACCAGTCCAGCAGCCGCTTTTATGTT GTGATGTGGAAGCAAGTCACCCAGTCCTACTGGGACACCAACCCCACGAGG GCTCAGGGATACTCGGGCCTTTCTGTGAAAGTTGTAAACTCCACCACAGGG CCTGGCGAGCACCTGCGGAACGCCCTGTGGCACACAGGAAACACCCCTGGC CAGGTGCGCACCCTGTGGCATGACCCTCGTCACATAGGCTGGAAAGATTTC ACCGCCTACAGATGGCGTCTCAGCCACAGGCCAAAGACGGGTTTCATTAGA GTGGTGATGTATGAAGGGAAGAAAATCATGGCTGACTCAGGACCCATCTAT GATAAAACCTATGCTGGTGGTAGACTAGGGTTGTTTGTCTTCTCTCAAGAA ATGGTGTTCTTCTCTGACCTGAAATACGAATGTAGAGATCCCTAA CO1A1_HUMAN ATGTTCAGCTTTGTGGACCTCCGGCTCCTGCTCCTCTTAGCGGCCACCGCC 6 CTCCTGACGCACGGCCAAGAGGAAGGCCAAGTCGAGGGCCAAGACGAAGAC ATCCCACCAATCACCTGCGTACAGAACGGCCTCAGGTACCATGACCGAGAC GTGTGGAAACCCGAGCCCTGCCGGATCTGCGTCTGCGACAACGGCAAGGTG TTGTGCGATGACGTGATCTGTGACGAGACCAAGAACTGCCCCGGCGCCGAA GTCCCCGAGGGCGAGTGCTGTCCCGTCTGCCCCGACGGCTCAGAGTCACCC ACCGACCAAGAAACCACCGGCGTCGAGGGACCCAAGGGAGACACTGGCCCC CGAGGCCCAAGGGGACCCGCAGGCCCCCCTGGCCGAGATGGCATCCCTGGA CAGCCTGGACTTCCCGGACCCCCCGGACCCCCCGGACCTCCCGGACCCCCT GGCCTCGGAGGAAACTTTGCTCCCCAGCTGTCTTATGGCTATGATGAGAAA TCAACCGGAGGAATTTCCGTGCCTGGCCCCATGGGTCCCTCTGGTCCTCGT GGTCTCCCTGGCCCCCCTGGTGCACCTGGTCCCCAAGGCTTCCAAGGTCCC CCTGGTGAGCCTGGCGAGCCTGGAGCTTCAGGTCCCATGGGTCCCCGAGGT CCCCCAGGTCCCCCTGGAAAGAATGGAGATGATGGGGAAGCTGGAAAACCT GGTCGTCCTGGTGAGCGTGGGCCTCCTGGGCCTCAGGGTGCTCGAGGATTG CCCGGAACAGCTGGCCTCCCTGGAATGAAGGGACACAGAGGTTTCAGTGGT TTGGATGGTGCCAAGGGAGATGCTGGTCCTGCTGGTCCTAAGGGTGAGCCT GGCAGCCCTGGTGAAAATGGAGCTCCTGGTCAGATGGGCCCCCGTGGCCTG CCTGGTGAGAGAGGTCGCCCTGGAGCCCCTGGCCCTGCTGGTGCTCGTGGA AATGATGGTGCTACTGGTGCTGCCGGGCCCCCTGGTCCCACCGGCCCCGCT GGTCCTCCTGGCTTCCCTGGTGCTGTTGGTGCTAAGGGTGAAGCTGGTCCC CAAGGGCCCCGAGGCTCTGAAGGTCCCCAGGGTGTGCGTGGTGAGCCTGGC CCCCCTGGCCCTGCTGGTGCTGCTGGCCCTGCTGGAAACCCTGGTGCTGAT GGACAGCCTGGTGCTAAAGGTGCCAATGGTGCTCCTGGTATTGCTGGTGCT CCTGGCTTCCCTGGTGCCCGAGGCCCCTCTGGACCCCAGGGCCCCGGCGGC CCTCCTGGTCCCAAGGGTAACAGCGGTGAACCTGGTGCTCCTGGCAGCAAA GGAGACACTGGTGCTAAGGGAGAGCCTGGCCCTGTTGGTGTTCAAGGACCC CCTGGCCCTGCTGGAGAGGAAGGAAAGCGAGGAGCTCGAGGTGAACCCGGA CCCACTGGCCTGCCCGGACCCCCTGGCGAGCGTGGTGGACCTGGTAGCCGT GGTTTCCCTGGCGCAGATGGTGTTGCTGGTCCCAAGGGTCCCGCTGGTGAA CGTGGTTCTCCTGGCCCTGCTGGCCCCAAAGGATCTCCTGGTGAAGCTGGT CGTCCCGGTGAAGCTGGTCTGCCTGGTGCCAAGGGTCTGACTGGAAGCCCT GGCAGCCCTGGTCCTGATGGCAAAACTGGCCCCCCTGGTCCCGCCGGTCAA GATGGTCGCCCCGGACCCCCAGGCCCACCTGGTGCCCGTGGTCAGGCTGGT GTGATGGGATTCCCTGGACCTAAAGGTGCTGCTGGAGAGCCCGGCAAGGCT GGAGAGCGAGGTGTTCCCGGACCCCCTGGCGCTGTCGGTCCTGCTGGCAAA GATGGAGAGGCTGGAGCTCAGGGACCCCCTGGCCCTGCTGGTCCCGCTGGC GAGAGAGGTGAACAAGGCCCTGCTGGCTCCCCCGGATTCCAGGGTCTCCCT GGTCCTGCTGGTCCTCCAGGTGAAGCAGGCAAACCTGGTGAACAGGGTGTT CCTGGAGACCTTGGCGCCCCTGGCCCCTCTGGAGCAAGAGGCGAGAGAGGT TTCCCTGGCGAGCGTGGTGTGCAAGGTCCCCCTGGTCCTGCTGGTCCCCGA GGGGCCAACGGTGCTCCCGGCAACGATGGTGCTAAGGGTGATGCTGGTGCC CCTGGAGCTCCCGGTAGCCAGGGCGCCCCTGGCCTTCAGGGAATGCCTGGT GAACGTGGTGCAGCTGGTCTTCCAGGGCCTAAGGGTGACAGAGGTGATGCT GGTCCCAAAGGTGCTGATGGCTCTCCTGGCAAAGATGGCGTCCGTGGTCTG ACTGGCCCCATTGGTCCTCCTGGCCCTGCTGGTGCCCCTGGTGACAAGGGT GAAAGTGGTCCCAGCGGCCCTGCTGGTCCCACTGGAGCTCGTGGTGCCCCC GGAGACCGTGGTGAGCCTGGTCCCCCCGGCCCTGCTGGCTTTGCTGGCCCC CCTGGTGCTGACGGCCAACCTGGTGCTAAAGGCGAACCTGGTGATGCTGGT GCTAAAGGCGATGCTGGTCCCCCTGGCCCTGCCGGACCCGCTGGACCCCCT GGCCCCATTGGTAATGTTGGTGCTCCTGGAGCCAAAGGTGCTCGCGGCAGC GCTGGTCCCCCTGGTGCTACTGGTTTCCCTGGTGCTGCTGGCCGAGTCGGT CCTCCTGGCCCCTCTGGAAATGCTGGACCCCCTGGCCCTCCTGGTCCTGCT GGCAAAGAAGGCGGCAAAGGTCCCCGTGGTGAGACTGGCCCTGCTGGACGT CCTGGTGAAGTTGGTCCCCCTGGTCCCCCTGGCCCTGCTGGCGAGAAAGGA TCCCCTGGTGCTGATGGTCCTGCTGGTGCTCCTGGTACTCCCGGGCCTCAA GGTATTGCTGGACAGCGTGGTGTGGTCGGCCTGCCTGGTCAGAGAGGAGAG AGAGGCTTCCCTGGTCTTCCTGGCCCCTCTGGTGAACCTGGCAAACAAGGT CCCTCTGGAGCAAGTGGTGAACGTGGTCCCCCTGGTCCCATGGGCCCCCCT GGATTGGCTGGACCCCCTGGTGAATCTGGACGTGAGGGGGCTCCTGGTGCC GAAGGTTCCCCTGGACGAGACGGTTCTCCTGGCGCCAAGGGTGACCGTGGT GAGACCGGCCCCGCTGGACCCCCTGGTGCTCCTGGTGCTCCTGGTGCCCCT GGCCCCGTTGGCCCTGCTGGCAAGAGTGGTGATCGTGGTGAGACTGGTCCT GCTGGTCCCACCGGTCCTGTCGGCCCTGTTGGCGCCCGTGGCCCCGCCGGA CCCCAAGGCCCCCGTGGTGACAAGGGTGAGACAGGCGAACAGGGCGACAGA GGCATAAAGGGTCACCGTGGCTTCTCTGGCCTCCAGGGTCCCCCTGGCCCT CCTGGCTCTCCTGGTGAACAAGGTCCCTCTGGAGCCTCTGGTCCTGCTGGT CCCCGAGGTCCCCCTGGCTCTGCTGGTGCTCCTGGCAAAGATGGACTCAAC GGTCTCCCTGGCCCCATTGGGCCCCCTGGTCCTCGCGGTCGCACTGGTGAT GCTGGTCCTGTTGGTCCCCCCGGCCCTCCTGGACCTCCTGGTCCCCCTGGT CCTCCCAGCGCTGGTTTCGACTTCAGCTTCCTGCCCCAGCCACCTCAAGAG AAGGCTCACGATGGTGGCCGCTACTACCGGGCTGATGATGCCAATGTGGTT CGTGACCGTGACCTCGAGGTGGACACCACCCTCAAGAGCCTGAGCCAGCAG ATCGAGAACATCCGGAGCCCAGAGGGCAGCCGCAAGAACCCCGCCCGCACC TGCCGTGACCTCAAGATGTGCCACTCTGACTGGAAGAGTGGAGAGTACTGG ATTGACCCCAACCAAGGCTGCAACCTGGATGCCATCAAAGTCTTCTGCAAC ATGGAGACTGGTGAGACCTGCGTGTACCCCACTCAGCCCAGTGTGGCCCAG AAGAACTGGTACATCAGCAAGAACCCCAAGGACAAGAGGCATGTCTGGTTC GGCGAGAGCATGACCGATGGATTCCAGTTCGAGTATGGCGGCCAGGGCTCC GACCCTGCCGATGTGGCCATCCAGCTGACCTTCCTGCGCCTGATGTCCACC GAGGCCTCCCAGAACATCACCTACCACTGCAAGAACAGCGTGGCCTACATG GACCAGCAGACTGGCAACCTCAAGAAGGCCCTGCTCCTCCAGGGCTCCAAC GAGATCGAGATCCGCGCCGAGGGCAACAGCCGCTTCACCTACAGCGTCACT GTCGATGGCTGCACGAGTCACACCGGAGCCTGGGGCAAGACAGTGATTGAA TACAAAACCACCAAGACCTCCCGCCTGCCCATCATCGATGTGGCCCCCTTG GACGTTGGTGCCCCAGACCAGGAATTCGGCTTCGACGTTGGCCCTGTCTGC TTCCTGTAA

TABLE 7 Amino acid sequences of proteins in high performing panels. Protein Seq. Name Amino Acid Sequence ID. ALDOA_HUMAN MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTEN 7 TEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKG GVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLK IGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQY VTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVT ALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRAL QASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAASESL FVSNHAY ALDOA_HUMAN MARRKPEGSSFKMTHLSMAMAFSFPPVASGQLHPQLGNTQHQTELGKELAT 8 (isoform TSTMPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIG 2) TENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIK SKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRC VLKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKR CQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMA TVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYG RALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAAS ESLFVSNHAY FRIL_HUMAN MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHF 9 FRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMA LEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLH RLGGPEAGLGEYLFERLTLKHD LG3BP_HUMAN MTPPRLFWVWLLVAGTQGVNDGDMRLADGGATNQGRVEIFYRGQWGTVCDN 10 LWDLTDASVVCRALGFENATQALGRAAFGQGSGPIMLDEVQCTGTEASLAD CKSLGWLKSNCRHERDAGVVCTNETRSTHTLDLSRELSEALGQIFDSQRGC DLSISVNVQGEDALGFCGHTVILTANLEAQALWREPGSNVTMSVDAECVPM VRDLLRYFYSRRIDITLSSVKCFHKLASAYGARQLQGYCASLFAILLPQDP SFQMPLDLYAYAVATGDALLEKLCLQFLAWNFEALTQAEAWPSVPTDLLQL LLPRSDLAVPSELALLKAVDTWSWGERASHEEVEGLVEKIRFPMMLPEELF ELQFNLSLYWSHEALFQKKTLQALEFHTVPFQLLARYKGLNLTEDTYKPRI YTSPTWSAFVTDSSWSARKSQLVYQSRRGPLVKYSSDYFQAPSDYRYYPYQ SFQTPQHPSFLFQDKRVSWSLVYLPTIQSCWNYGFSCSSDELPVLGLTKSG GSDRTIAYENKALMLCEGLFVADVTDFEGWKAAIPSALDTNSSKSTSSFPC PAGHFNGFRTVIRPFYLTNSSGVD TSP1_HUMAN MGLAWGLGVLFLMHVCGTNRIPESGGDNSVFDIFELTGAARKGSGRRLVKG 11 PDPSSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKGFLLLASLRQMKKTRG TLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEEALLATGQW KSITLFVQEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIAKGGV NDNFQGVLQNVRFVFGTTPEDILRNKGCSSSTSVLLTLDNNVVNGSSPAIR TNYIGHKTKDLQAICGISCDELSSMVLELRGLRTIVTTLQDSIRKVTEENK ELANELRRPPLCYHNGVQYRNNEEWTVDSCTECHCQNSVTICKKVSCPIMP CSNATVPDGECCPRCWPSDSADDGWSPWSEWTSCSTSCGNGIQQRGRSCDS LNNRCEGSSVQTRTCHIQECDKRFKQDGGWSHWSPWSSCSVTCGDGVITRI RLCNSPSPQMNGKPCEGEARETKACKKDACPINGGWGPWSPWDICSVTCGG GVQKRSRLCNNPTPQFGGKDCVGDVTENQICNKQDCPIDGCLSNPCFAGVK CTSYPDGSWKCGACPPGYSGNGIQCTDVDECKEVPDACFNHNGEHRCENTD PGYNCLPCPPRFTGSQPFGQGVEHATANKQVCKPRNPCTDGTHDCNKNAKC NYLGHYSDPMYRCECKPGYAGNGIICGEDTDLDGWPNENLVCVANATYHCK KDNCPNLPNSGQEDYDKDGIGDACDDDDDNDKIPDDRDNCPFHYNPAQYDY DRDDVGDRCDNCPYNHNPDQADTDNNGEGDACAADIDGDGILNERDNCQYV YNVDQRDTDMDGVGDQCDNCPLEHNPDQLDSDSDRIGDTCDNNQDIDEDGH QNNLDNCPYVPNANQADHDKDGKGDACDHDDDNDGIPDDKDNCRLVPNPDQ KDSDGDGRGDACKDDFDHDSVPDIDDICPENVDISETDFRRFQMIPLDPKG TSQNDPNWVVRHQGKELVQTVNCDPGLAVGYDEFNAVDFSGTFFINTERDD DYAGFVFGYQSSSRFYVVMWKQVTQSYWDTNPTRAQGYSGLSVKVVNSTTG PGEHLRNALWHTGNTPGQVRTLWHDPRHIGWKDFTAYRWRLSHRPKTGFIR VVMYEGKKIMADSGPIYDKTYAGGRLGLFVFSQEMVFFSDLKYECRDP CO1A1_HUMAN MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRD 12 VWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESP TDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPP GLGGNFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGP PGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGL PGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGL PGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGP QGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGA PGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGP PGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGE RGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQ DGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGK DGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGV PGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGA PGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGL TGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGP PGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGS AGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGR PGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGE RGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGA EGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGP AGPTGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGP PGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGD AGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVV RDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYW IDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWF GESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYM DQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIE YKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL

TABLE 8 Nucleotide sequences of normalizer proteins in panel. Seq. Gene Name Nucleotide Sequence ID. PEDF_HUMAN ATGCAGGCCCTGGTGCTACTCCTCTGCATTGGAGCCCTCCTCGGGCACAGC 13 AGCTGCCAGAACCCTGCCAGCCCCCCGGAGGAGGGCTCCCCAGACCCCGAC AGCACAGGGGCGCTGGTGGAGGAGGAGGATCCTTTCTTCAAAGTCCCCGTG AACAAGCTGGCAGCGGCTGTCTCCAACTTCGGCTATGACCTGTACCGGGTG CGATCCAGCACGAGCCCCACGACCAACGTGCTCCTGTCTCCTCTCAGTGTG GCCACGGCCCTCTCGGCCCTCTCGCTGGGAGCGGAGCAGCGAACAGAATCC ATCATTCACCGGGCTCTCTACTATGACTTGATCAGCAGCCCAGACATCCAT GGTACCTATAAGGAGCTCCTTGACACGGTCACTGCCCCCCAGAAGAACCTC AAGAGTGCCTCCCGGATCGTCTTTGAGAAGAAGCTGCGCATAAAATCCAGC TTTGTGGCACCTCTGGAAAAGTCATATGGGACCAGGCCCAGAGTCCTGACG GGCAACCCTCGCTTGGACCTGCAAGAGATCAACAACTGGGTGCAGGCGCAG ATGAAAGGGAAGCTCGCCAGGTCCACAAAGGAAATTCCCGATGAGATCAGC ATTCTCCTTCTCGGTGTGGCGCACTTCAAGGGGCAGTGGGTAACAAAGTTT GACTCCAGAAAGACTTCCCTCGAGGATTTCTACTTGGATGAAGAGAGGACC GTGAGGGTCCCCATGATGTCGGACCCTAAGGCTGTTTTACGCTATGGCTTG GATTCAGATCTCAGCTGCAAGATTGCCCAGCTGCCCTTGACCGGAAGCATG AGTATCATCTTCTTCCTGCCCCTGAAAGTGACCCAGAATTTGACCTTGATA GAGGAGAGCCTCACCTCCGAGTTCATTCATGACATAGACCGAGAACTGAAG ACCGTGCAGGCGGTCCTCACTGTCCCCAAGCTGAAGCTGAGTTATGAAGGC GAAGTCACCAAGTCCCTGCAGGAGATGAAGCTGCAATCCTTGTTTGATTCA CCAGACTTTAGCAAGATCACAGGCAAACCCATCAAGCTGACTCAGGTGGAA CACCGGGCTGGCTTTGAGTGGAACGAGGATGGGGCGGGAACCACCCCCAGC CCAGGGCTGCAGCCTGCCCACCTCACCTTCCCGCTGGACTATCACCTTAAC CAGCCTTTCATCTTCGTACTGAGGGACACAGACACAGGGGCCCTTCTCTTC ATTGGCAAGATTCTGGACCCCAGGGGCCCCTAA MASP1_HUMAN ATGAGGTGGCTGCTTCTCTATTATGCTCTGTGCTTCTCCCTGTCAAAGGCT 14 TCAGCCCACACCGTGGAGCTAAACAATATGTTTGGCCAGATCCAGTCGCCT GGTTATCCAGACTCCTATCCCAGTGATTCAGAGGTGACTTGGAATATCACT GTCCCAGATGGGTTTCGGATCAAGCTTTACTTCATGCACTTCAACTTGGAA TCCTCCTACCTTTGTGAATATGACTATGTGAAGGTAGAAACTGAGGACCAG GTGCTGGCAACCTTCTGTGGCAGGGAGACCACAGACACAGAGCAGACTCCC GGCCAGGAGGTGGTCCTCTCCCCTGGCTCCTTCATGTCCATCACTTTCCGG TCAGATTTCTCCAATGAGGAGCGTTTCACAGGCTTTGATGCCCACTACATG GCTGTGGATGTGGACGAGTGCAAGGAGAGGGAGGACGAGGAGCTGTCCTGT GACCACTACTGCCACAACTACATTGGCGGCTACTACTGCTCCTGCCGCTTC GGCTACATCCTCCACACAGACAACAGGACCTGCCGAGTGGAGTGCAGTGAC AACCTCTTCACTCAAAGGACTGGGGTGATCACCAGCCCTGACTTCCCAAAC CCTTACCCCAAGAGCTCTGAATGCCTGTATACCATCGAGCTGGAGGAGGGT TTCATGGTCAACCTGCAGTTTGAGGACATATTTGACATTGAGGACCATCCT GAGGTGCCCTGCCCCTATGACTACATCAAGATCAAAGTTGGTCCAAAAGTT TTGGGGCCTTTCTGTGGAGAGAAAGCCCCAGAACCCATCAGCACCCAGAGC CACAGTGTCCTGATCCTGTTCCATAGTGACAACTCGGGAGAGAACCGGGGC TGGAGGCTCTCATACAGGGCTGCAGGAAATGAGTGCCCAGAGCTACAGCCT CCTGTCCATGGGAAAATCGAGCCCTCCCAAGCCAAGTATTTCTTCAAAGAC CAAGTGCTCGTCAGCTGTGACACAGGCTACAAAGTGCTGAAGGATAATGTG GAGATGGACACATTCCAGATTGAGTGTCTGAAGGATGGGACGTGGAGTAAC AAGATTCCCACCTGTAAAATTGTAGACTGTAGAGCCCCAGGAGAGCTGGAA CACGGGCTGATCACCTTCTCTACAAGGAACAACCTCACCACATACAAGTCT GAGATCAAATACTCCTGTCAGGAGCCCTATTACAAGATGCTCAACAATAAC ACAGGTATATATACCTGTTCTGCCCAAGGAGTCTGGATGAATAAAGTATTG GGGAGAAGCCTACCCACCTGCCTTCCAGTGTGTGGGCTCCCCAAGTTCTCC CGGAAGCTGATGGCCAGGATCTTCAATGGACGCCCAGCCCAGAAAGGCACC ACTCCCTGGATTGCCATGCTGTCACACCTGAATGGGCAGCCCTTCTGCGGA GGCTCCCTTCTAGGCTCCAGCTGGATCGTGACCGCCGCACACTGCCTCCAC CAGTCACTCGATCCGGAAGATCCGACCCTACGTGATTCAGACTTGCTCAGC CCTTCTGACTTCAAAATCATCCTGGGCAAGCATTGGAGGCTCCGGTCAGAT GAAAATGAACAGCATCTCGGCGTCAAACACACCACTCTCCACCCCCAGTAT GATCCCAACACATTCGAGAATGACGTGGCTCTGGTGGAGCTGTTGGAGAGC CCAGTGCTGAATGCCTTCGTGATGCCCATCTGTCTGCCTGAGGGACCCCAG CAGGAAGGAGCCATGGTCATCGTCAGCGGCTGGGGGAAGCAGTTCTTGCAA AGGTTCCCAGAGACCCTGATGGAGATTGAAATCCCGATTGTTGACCACAGC ACCTGCCAGAAGGCTTATGCCCCGCTGAAGAAGAAAGTGACCAGGGACATG ATCTGTGCTGGGGAGAAGGAAGGGGGAAAGGACGCCTGTGCGGGTGACTCT GGAGGCCCCATGGTGACCCTGAATAGAGAAAGAGGCCAGTGGTACCTGGTG GGCACTGTGTCCTGGGGTGATGACTGTGGGAAGAAGGACCGCTACGGAGTA TACTCTTACATCCACCACAACAAGGACTGGATCCAGAGGGTCACCGGAGTG AGGAACTGA GELS_HUMAN ATGGCTCCGCACCGCCCCGCGCCCGCGCTGCTTTGCGCGCTGTCCCTGGCG 15 CTGTGCGCGCTGTCGCTGCCCGTCCGCGCGGCCACTGCGTCGCGGGGGGCG TCCCAGGCGGGGGCGCCCCAGGGGCGGGTGCCCGAGGCGCGGCCCAACAGC ATGGTGGTGGAACACCCCGAGTTCCTCAAGGCAGGGAAGGAGCCTGGCCTG CAGATCTGGCGTGTGGAGAAGTTCGATCTGGTGCCCGTGCCCACCAACCTT TATGGAGACTTCTTCACGGGCGACGCCTACGTCATCCTGAAGACAGTGCAG CTGAGGAACGGAAATCTGCAGTATGACCTCCACTACTGGCTGGGCAATGAG TGCAGCCAGGATGAGAGCGGGGCGGCCGCCATCTTTACCGTGCAGCTGGAT GACTACCTGAACGGCCGGGCCGTGCAGCACCGTGAGGTCCAGGGCTTCGAG TCGGCCACCTTCCTAGGCTACTTCAAGTCTGGCCTGAAGTACAAGAAAGGA GGTGTGGCATCAGGATTCAAGCACGTGGTACCCAACGAGGTGGTGGTGCAG AGACTCTTCCAGGTCAAAGGGCGGCGTGTGGTCCGTGCCACCGAGGTACCT GTGTCCTGGGAGAGCTTCAACAATGGCGACTGCTTCATCCTGGACCTGGGC AACAACATCCACCAGTGGTGTGGTTCCAACAGCAATCGGTATGAAAGACTG AAGGCCACACAGGTGTCCAAGGGCATCCGGGACAACGAGCGGAGTGGCCGG GCCCGAGTGCACGTGTCTGAGGAGGGCACTGAGCCCGAGGCGATGCTCCAG GTGCTGGGCCCCAAGCCGGCTCTGCCTGCAGGTACCGAGGACACCGCCAAG GAGGATGCGGCCAACCGCAAGCTGGCCAAGCTCTACAAGGTCTCCAATGGT GCAGGGACCATGTCCGTCTCCCTCGTGGCTGATGAGAACCCCTTCGCCCAG GGGGCCCTGAAGTCAGAGGACTGCTTCATCCTGGACCACGGCAAAGATGGG AAAATCTTTGTCTGGAAAGGCAAGCAGGCAAACACGGAGGAGAGGAAGGCT GCCCTCAAAACAGCCTCTGACTTCATCACCAAGATGGACTACCCCAAGCAG ACTCAGGTCTCGGTCCTTCCTGAGGGCGGTGAGACCCCACTGTTCAAGCAG TTCTTCAAGAACTGGCGGGACCCAGACCAGACAGATGGCCTGGGCTTGTCC TACCTTTCCAGCCATATCGCCAACGTGGAGCGGGTGCCCTTCGACGCCGCC ACCCTGCACACCTCCACTGCCATGGCCGCCCAGCACGGCATGGATGACGAT GGCACAGGCCAGAAACAGATCTGGAGAATCGAAGGTTCCAACAAGGTGCCC GTGGACCCTGCCACATATGGACAGTTCTATGGAGGCGACAGCTACATCATT CTGTACAACTACCGCCATGGTGGCCGCCAGGGGCAGATAATCTATAACTGG CAGGGTGCCCAGTCTACCCAGGATGAGGTCGCTGCATCTGCCATCCTGACT GCTCAGCTGGATGAGGAGCTGGGAGGTACCCCTGTCCAGAGCCGTGTGGTC CAAGGCAAGGAGCCCGCCCACCTCATGAGCCTGTTTGGTGGGAAGCCCATG ATCATCTACAAGGGCGGCACCTCCCGCGAGGGCGGGCAGACAGCCCCTGCC AGCACCCGCCTCTTCCAGGTCCGCGCCAACAGCGCTGGAGCCACCCGGGCT GTTGAGGTATTGCCTAAGGCTGGTGCACTGAACTCCAACGATGCCTTTGTT CTGAAAACCCCCTCAGCCGCCTACCTGTGGGTGGGTACAGGAGCCAGCGAG GCAGAGAAGACGGGGGCCCAGGAGCTGCTCAGGGTGCTGCGGGCCCAACCT GTGCAGGTGGCAGAAGGCAGCGAGCCAGATGGCTTCTGGGAGGCCCTGGGC GGGAAGGCTGCCTACCGCACATCCCCACGGCTGAAGGACAAGAAGATGGAT GCCCATCCTCCTCGCCTCTTTGCCTGCTCCAACAAGATTGGACGTTTTGTG ATCGAAGAGGTTCCTGGTGAGCTCATGCAGGAAGACCTGGCAACGGATGAC GTCATGCTTCTGGACACCTGGGACCAGGTCTTTGTCTGGGTTGGAAAGGAT TCTCAAGAAGAAGAAAAGACAGAAGCCTTGACTTCTGCTAAGCGGTACATC GAGACGGACCCAGCCAATCGGGATCGGCGGACGCCCATCACCGTGGTGAAG CAAGGCTTTGAGCCTCCCTCCTTTGTGGGCTGGTTCCTTGGCTGGGATGAT GATTACTGGTCTGTGGACCCCTTGGACAGGGCCATGGCTGAGCTGGCTGCC TGA LUM_HUMAN ATGAGTCTAAGTGCATTTACTCTCTTCCTGGCATTGATTGGTGGTACCAGT 16 GGCCAGTACTATGATTATGATTTTCCCCTATCAATTTATGGGCAATCATCA CCAAACTGTGCACCAGAATGTAACTGCCCTGAAAGCTACCCAAGTGCCATG TACTGTGATGAGCTGAAATTGAAAAGTGTACCAATGGTGCCTCCTGGAATC AAGTATCTTTACCTTAGGAATAACCAGATTGACCATATTGATGAAAAGGCC TTTGAGAATGTAACTGATCTGCAGTGGCTCATTCTAGATCACAACCTTCTA GAAAACTCCAAGATAAAAGGGAGAGTTTTCTCTAAATTGAAACAACTGAAG AAGCTGCATATAAACCACAACAACCTGACAGAGTCTGTGGGCCCACTTCCC AAATCTCTGGAGGATCTGCAGCTTACTCATAACAAGATCACAAAGCTGGGC TCTTTTGAAGGATTGGTAAACCTGACCTTCATCCATCTCCAGCACAATCGG CTGAAAGAGGATGCTGTTTCAGCTGCTTTTAAAGGTCTTAAATCACTCGAA TACCTTGACTTGAGCTTCAATCAGATAGCCAGACTGCCTTCTGGTCTCCCT GTCTCTCTTCTAACTCTCTACTTAGACAACAATAAGATCAGCAACATCCCT GATGAGTATTTCAAGCGTTTTAATGCATTGCAGTATCTGCGTTTATCTCAC AACGAACTGGCTGATAGTGGAATACCTGGAAATTCTTTCAATGTGTCATCC CTGGTTGAGCTGGATCTGTCCTATAACAAGCTTAAAAACATACCAACTGTC AATGAAAACCTTGAAAACTATTACCTGGAGGTCAATCAACTTGAGAAGTTT GACATAAAGAGCTTCTGCAAGATCCTGGGGCCATTATCCTACTCCAAGATC AAGCATTTGCGTTTGGATGGCAATCGCATCTCAGAAACCAGTCTTCCACCG GATATGTATGAATGTCTACGTGTTGCTAACGAAGTCACTCTTAATTAA C163A_HUMAN ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACTTC 17 AGAAGACATTTTGTCAACTTGAGTCCCTTCACCATTACTGTGGTCTTACTT CTCAGTGCCTGTTTTGTCACCAGTTCTCTTGGAGGAACAGACAAGGAGCTG AGGCTAGTGGATGGTGAAAACAAGTGTAGCGGGAGAGTGGAAGTGAAAGTC CAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGGAGCATGGAAGCGGTC TCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCCCCTGGA TGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCT TGTCGTGGGAATGAGTCAGCTCTTTGGGATTGCAAACATGATGGATGGGGA AAGCATAGTAACTGTACTCACCAACAAGATGCTGGAGTGACCTGCTCAGAT GGATCCAATTTGGAAATGAGGCTGACGCGTGGAGGGAATATGTGTTCTGGA AGAATAGAGATCAAATTCCAAGGACGGTGGGGAACAGTGTGTGATGATAAC TTCAACATAGATCATGCATCTGTCATTTGTAGACAACTTGAATGTGGAAGT GCTGTCAGTTTCTCTGGTTCATCTAATTTTGGAGAAGGCTCTGGACCAATC TGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGC AAACATCAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGA GTGATTTGCTCAAAGGGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTC ACTGAATGTTCAGGAAGATTAGAAGTGAGATTCCAAGGAGAATGGGGGACA ATATGTGATGACGGCTGGGACAGTTACGATGCTGCTGTGGCATGCAAGCAA CTGGGATGTCCAACTGCCGTCACAGCCATTGGTCGAGTTAACGCCAGTAAG GGATTTGGACACATCTGGCTTGACAGCGTTTCTTGCCAGGGACATGAACCT GCTATCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCAC AATGAAGATGCTGGCGTGACATGTTCTGATGGATCAGATCTGGAGCTAAGA CTTAGAGGTGGAGGCAGCCGCTGTGCTGGGACAGTTGAGGTGGAGATTCAG AGACTGTTAGGGAAGGTGTGTGACAGAGGCTGGGGACTGAAAGAAGCTGAT GTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTCAAAACATCTTATCAA GTGTACTCCAAAATCCAGGCAACAAACACATGGCTGTTTCTAAGTAGCTGT AACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGA CTTACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGG GAACCCAGACTGGTTGGAGGGGACATTCCCTGTTCTGGACGTGTTGAAGTG AAGCATGGTGACACGTGGGGCTCCATCTGTGATTCGGACTTCTCTCTGGAA GCTGCCAGCGTTCTATGCAGGGAATTACAGTGTGGCACAGTTGTCTCTATC CTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATCTGGGCTGAAGAA TTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCACCC CGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCA AGATACACAGAAATTCGCTTGGTGAATGGCAAGACCCCGTGTGAGGGCAGA GTGGAGCTCAAAACGCTTGGTGCCTGGGGATCCCTCTGTAACTCTCACTGG GACATAGAAGATGCCCATGTTCTTTGCCAGCAGCTTAAATGTGGAGTTGCC CTTTCTACCCCAGGAGGAGCACGTTTTGGAAAAGGAAATGGTCAGATCTGG AGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGAGATTGTCCT GTAACTGCTCTAGGTGCTTCATTATGTCCTTCAGAGCAAGTGGCCTCTGTA ATCTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCGTCT TTGGGCCCAACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATA GAGAGTGGTCAACTTCGCCTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGA GTAGAGATCTATCATGAGGGCTCCTGGGGCACCATCTGTGATGACAGCTGG GACCTGAGTGATGCCCACGTGGTTTGCAGACAGCTGGGCTGTGGAGAGGCC ATTAATGCCACTGGTTCTGCTCATTTTGGGGAAGGAACAGGGCCCATCTGG CTGGATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCAT TCACACGGCTGGGGGCAGCAAAATTGCAGGCACAAGGAGGATGCGGGAGTT ATCTGCTCAGAATTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAG GCCTGTGCAGGGCGTCTGGAAGTTTTTTACAATGGAGCTTGGGGCACTGTT GGCAAGAGTAGCATGTCTGAAACCACTGTGGGTGTGGTGTGCAGGCAGCTG GGCTGTGCAGACAAAGGGAAAATCAACCCTGCATCTTTAGACAAGGCCATG TCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGACACG CTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGCCCCTCG GAGGAGACCTGGATCACATGTGACAACAAGATAAGACTTCAGGAAGGACCC ACTTCCTGTTCTGGACGTGTGGAGATCTGGCATGGAGGTTCCTGGGGGACA GTGTGTGATGACTCTTGGGACTTGGACGATGCTCAGGTGGTGTGTCAACAA CTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAAGAAGCAGAGTTTGGTCAG GGGACTGGACCGATATGGCTCAATGAAGTGAAGTGCAAAGGGAATGAGTCT TCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCATAGTGAGTGTGGGCAC AAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAGTGCAGAAAACCCCA CAAAAAGCCACAACAGGTCGCTCATCCCGTCAGTCATCCTTTATTGCAGTC GGGATCCTTGGGGTTGTTCTGTTGGCCATTTTCGTCGCATTATTCTTCTTG ACTAAAAAGCGAAGACAGAGACAGCGGCTTGCAGTTTCCTCAAGAGGAGAG AACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTGAATGCA GATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCTGAGCCACACTGA PTPRJ_HUMAN ATGAAGCCGGCGGCGCGGGAGGCGCGGCTGCCTCCGCGCTCGCCCGGGCTG 18 CGCTGGGCGCTGCCGCTGCTGCTGCTGCTGCTGCGCCTGGGCCAGATCCTG TGCGCAGGTGGCACCCCTAGTCCAATTCCTGACCCTTCAGTAGCAACTGTT GCCACAGGGGAAAATGGCATAACGCAGATCAGCAGTACAGCAGAATCCTTT CATAAACAGAATGGAACTGGAACACCTCAGGTGGAAACAAACACCAGTGAG GATGGTGAAAGCTCTGGAGCCAACGATAGTTTAAGAACACCTGAACAAGGA TCTAATGGGACTGATGGGGCATCTCAAAAAACTCCCAGTAGCACTGGGCCC AGTCCTGTGTTTGACATTAAAGCTGTTTCCATCAGTCCAACCAATGTGATC TTAACTTGGAAAAGTAATGACACAGCTGCTTCTGAGTACAAGTATGTAGTA AAGCATAAGATGGAAAATGAGAAGACAATTACTGTTGTGCATCAACCATGG TGTAACATCACAGGCTTACGTCCAGCGACTTCATATGTATTCTCCATCACT CCAGGAATAGGCAATGAGACTTGGGGAGATCCCAGAGTCATAAAAGTCATC ACAGAGCCGATCCCAGTTTCTGATCTCCGTGTTGCCCTCACGGGTGTGAGG AAGGCTGCTCTCTCCTGGAGCAATGGCAATGGCACTGCCTCCTGCCGGGTT CTTCTTGAAAGCATTGGAAGCCATGAGGAGTTGACTCAAGACTCAAGACTT CAGGTCAATATCTCGGGCCTGAAGCCAGGGGTTCAATACAACATCAACCCG TATCTTCTACAATCAAATAAGACAAAGGGAGACCCCTTGGGCACAGAAGGT GGCTTGGATGCCAGCAATACAGAGAGAAGCCGGGCAGGGAGCCCCACCGCC CCTGTGCATGATGAGTCCCTCGTGGGACCTGTGGACCCATCCTCCGGCCAG CAGTCCCGAGACACGGAAGTCCTGCTTGTCGGGTTAGAGCCTGGCACCCGA TACAATGCCACCGTTTATTCCCAAGCAGCGAATGGCACAGAAGGACAGCCC CAGGCCATAGAGTTCAGGACAAATGCTATTCAGGTTTTTGACGTCACCGCT GTGAACATCAGTGCCACAAGCCTGACCCTGATCTGGAAAGTCAGCGATAAC GAGTCGTCATCTAACTATACCTACAAGATACATGTGGCGGGGGAGACAGAT TCTTCCAATCTCAACGTCAGTGAGCCTCGCGCTGTCATCCCCGGACTCCGC TCCAGCACCTTCTACAACATCACAGTGTGTCCTGTCCTAGGTGACATCGAG GGCACGCCGGGCTTCCTCCAAGTGCACACCCCCCCTGTTCCAGTTTCTGAC TTCCGAGTGACAGTGGTCAGCACGACGGAGATCGGCTTAGCATGGAGCAGC CATGATGCAGAATCATTTCAGATGCATATCACACAGGAGGGAGCTGGCAAT TCTCGGGTAGAAATAACCACCAACCAAAGTATTATCATTGGTGGCTTGTTC CCTGGAACCAAGTATTGCTTTGAAATAGTTCCAAAAGGACCAAATGGGACT GAAGGGGCATCTCGGACAGTTTGCAATAGAACTGGATGA

TABLE 9 Amino acid sequences of normalizer proteins in panel. Seq. Gene Name Nucleotide Sequence ID. PEDF_HUMAN MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPV 19 NKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTES IIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSS FVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEIS ILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGL DSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELK TVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVE HRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLF IGKILDPRGP MASP1_HUMAN MRWLLLYYALCFSLSKASAHTVELNNMFGQIQSPGYPDSYPSDSEVTWNIT 20 VPDGFRIKLYFMHFNLESSYLCEYDYVKVETEDQVLATFCGRETTDTEQTP GQEVVLSPGSFMSITFRSDFSNEERFTGFDAHYMAVDVDECKEREDEELSC DHYCHNYIGGYYCSCRFGYILHTDNRTCRVECSDNLFTQRTGVITSPDFPN PYPKSSECLYTIELEEGFMVNLQFEDIFDIEDHPEVPCPYDYIKIKVGPKV LGPFCGEKAPEPISTQSHSVLILFHSDNSGENRGWRLSYRAAGNECPELQP PVHGKIEPSQAKYFFKDQVLVSCDTGYKVLKDNVEMDTFQIECLKDGTWSN KIPTCKIVDCRAPGELEHGLITFSTRNNLTTYKSEIKYSCQEPYYKMLNNN TGIYTCSAQGVWMNKVLGRSLPTCLPVCGLPKFSRKLMARIFNGRPAQKGT TPWIAMLSHLNGQPFCGGSLLGSSWIVTAAHCLHQSLDPEDPTLRDSDLLS PSDFKIILGKHWRLRSDENEQHLGVKHTTLHPQYDPNTFENDVALVELLES PVLNAFVMPICLPEGPQQEGAMVIVSGWGKQFLQRFPETLMEIEIPIVDHS TCQKAYAPLKKKVTRDMICAGEKEGGKDACAGDSGGPMVTLNRERGQWYLV GTVSWGDDCGKKDRYGVYSYIHHNKDWIQPVTGVRN GELS_HUMAN MAPHRPAPALLCALSLALCALSLPVRAATASRGASQAGAPQGRVPEARPNS 21 MVVEHPEFLKAGKEPGLQIWRVEKFDLVPVPTNLYGDFFTGDAYVILKTVQ LRNGNLQYDLHYWLGNECSQDESGAAAIFTVQLDDYLNGRAVQHREVQGFE SATFLGYFKSGLKYKKGGVASGFKHVVPNEVVVQRLFQVKGRRVVRATEVP VSWESFNNGDCFILDLGNNIHQWCGSNSNRYERLKATQVSKGIPDNERSGR ARVHVSEEGTEPEAMLQVLGPKPALPAGTEDTAKEDAANRKLAKLTASDFI TKMDYPKQTQVSVLPEGGETPLFKQFFKNWRDPDQTDGLGLSYLSSHIANV ERVPFDAATLHTSTAMAAQHGMDDDGTGQKQIWRIEGSNKVPVDPATYGQF YGGDSYIILYNYRHGGRQGQIIYNWQGAQSTQDEVAASAILTAQLDEELGG TPVQSRVVQGKEPAHLMSLFGGKPMIIYKGGTSREGGQTAPASTRLFQVRA NSAGATRAVEVLPKAGALNSNDAFVLKTPSAAYLWVGTGASEAEKTGAQEL LPVLRAQPVQVAEGSEPDGFWEALGGKAAYRTSPKLKDKKMDAHPPRLFAC SNKIGRFVIEEVPGELMQEDLATDDVMLLDTWDQVFVWVGKDSQEEEKTEA LTSAKRYIETDPANRDRRTPITVVKQGFEPPSFVGWFLGWDDDYWSVDPLD RAMAELAA LUM_HUMAN MSLSAFTLFLALIGGTSGQYYDYDFPLSIYGQSSPNCAPECNCPESYPSAM 22 YCDELKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLL ENSKIKGRVFSKLKQLKKLHINHNNLTESVGPLPKSLEDLQLTHNKITKLG SFEGLVNLTFIHLQHNRLKEDAVSAAFKGLKSLEYLDLSFNQIARLPSGLP VSLLTLYLDNNKISNIPDEYFKRFNALQYLRLSHNELADSGIPGNSFNVSS LVELDLSYNKLKNIPTVNENLENYYLEVNQLEKFDIKSFCKILGPLSYSKI KHLRLDGNRISETSLPPDMYECLRVANEVTLN C163A_HUMAN MSKLRMVLLEDSGSADFRRHFVNLSPFTITVVLLLSACFVTSSLGGTDKEL 23 RLVDGENKCSGRVEVKVQEEWGTVCNNGWSMEAVSVICNQLGCPTAIKAPG WANSSAGSGRIWMDHVSCRGNESALWDCKHDGWGKHSNCTHQQDAGVTCSD GSNLEMRLTRGGNMCSGRIEIKFQGRWGTVCDDNFNIDHASVICRQLECGS AVSFSGSSNFGEGSGPIWFDDLICNGNESALWNCKHQGWGKHNCDHAEDAG VICSKGADLSLRLVDGVTECSGRLEVRFQGEWGTICDDGWDSYDAAVACKQ LGCPTAVTAIGRVNASKGFGHIWLDSVSCQGHEPAIWQCKHHEWGKHYCNH NEDAGVTCSDGSDLELRLRGGGSRCAGTVEVEIQRLLGKVCDRGWGLKEAD VVCRQLGCGSALKTSYQVYSKIQATNTWLFLSSCNGNETSLWDCKNWQWGG LTCDHYEEAKITCSAHREPRLVGGDIPCSGRVEVKHGDTWGSICDSDFSLE AASVLCRELQCGTVVSILGGAHFGEGNGQIWAEEFQCEGHESHLSLCPVAP RPEGTCSHSRDVGVVCSRYTEIRLVNGKTPCEGRVELKTLGAWGSLCNSHW DIEDAHVLCQQLKCGVALSTPGGARFGKGNGQIWRHMFHCTGTEQHMGDCP VTALGASLCPSEQVASVICSGNQSQTLSSCNSSSLGPTRPTIPEESAVACI ESGQLRLVNGGGRCAGRVEIYHEGSWGTICDDSWDLSDAHVVCRQLGCGEA INATGSAHFGEGTGPIWLDEMKCNGKESRIWQCHSHGWGQQNCRHKEDAGV ICSEFMSLRLTSEASREACAGRLEVFYNGAWGTVGKSSMSETTVGVVCRQL GCADKGKINPASLDKAMSIPMWVDNVQCPKGPDTLWQCPSSPWEKRLASPS EETWITCDNKIRLQEGPTSCSGRVEIWHGGSWGTVCDDSWDLDDAQVVCQQ LGCGPALKAFKEAEFGQGTGPIWLNEVKCKGNESSLWDCPARRWGHSECGH KEDAAVNCTDISVQKTPQKATTGRSSRQSSFIAVGILGVVLLAIFVALFFL TKKRRQRQRLAVSSRGENLVHQIQYREMNSCLNADDLDLMNSSENSHESAD FSAAELISVSKFLPISGMEKEAILSHTEKENGNL PTPRJ_HUMAN MKPAAREARLPPRSPGLRWALPLLLLLLRLGQILCAGGTPSPIPDPSVATV 24 ATGENGITQISSTAESFHKQNGTGTPQVETNTSEDGESSGANDSLRTPEQG SNGTDGASQKTPSSTGPSPVFDIKAVSISPTNVILTWKSNDTAASEYKYVV KHKMENEKTITVVHQPWCNITGLRPATSYVFSITPGIGNETWGDPRVIKVI TEPIPVSDLRVALTGVRKAALSWSNGNGTASCRVLLESIGSHEELTQDSRL QVNISGLKPGVQYNINPYLLQSNKTKGDPLGTEGGLDASNTERSRAGSPTA PVHDESLVGPVDPSSGQQSRDTEVLLVGLEPGTRYNATVYSQAANGTEGQP QAIEFRTNAIQVFDVTAVNISATSLTLIWKVSDNESSSNYTYKIHVAGETD SSNLNVSEPRAVIPGLRSSTFYNITVCPVLGDIEGTPGFLQVHTPPVPVSD FRVTVVSTTEIGLAWSSHDAESFQMHITQEGAGNSRVEITTNQSIIIGGLF PGTKYCFEIVPKGPNGTEGASRTVCNRTVPSAVFDIHVVYVTTTEMWLDWK SPDGASEYVYHLVIESKHGSNHTSTYDKAITLQGLIPGTLYNITISPEVDH VWGDPNSTAQYTRPSNVSNIDVSTNTTAATLSWQNFDDASPTYSYCLLIEK AGNSSNATQVVTDIGITDATVTELIPGSSYTVEIFAQVGDGIKSLEPGRKS FCTDPASMASFDCEVVPKEPALVLKWTCPPGANAGFELEVSSGAWNNATHL ESCSSENGTEYRTEVTYLNFSTSYNISITTVSCGKMAAPTRNTCTTGITDP PPPDGSPNITSVSHNSVKVKFSGFEASHGPIKAYAVILTTGEAGHPSADVL KYTYEDFKKGASDTYVTYLIRTEEKGRSQSLSEVLKYEIDVGNESTTLGYY NGKLEPLGSYRACVAGFTNITFHPQNKGLIDGAESYVSFSRYSDAVSLPQD PGVICGAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKL IRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRY NNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKD FWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIV LPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRD YMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLR MHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPV TTFGKTNGYIA 

What is claimed is:
 1. A method of determining the likelihood that a pulmonary nodule in a subject is not lung cancer, comprising: (a) contacting a blood sample obtained from the subject with a proteolytic enzyme to produce peptide fragments from a panel of proteins present in the blood sample, wherein the panel comprises ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN, COIA1_HUMAN, PEDF_HUMAN, MASP1_HUMAN, GELS_HUMAN, LUM_HUMAN, C163A_HUMAN and PTPRJ_HUMAN; (b) combining the produced peptide fragments from the panel from step (a) with labeled, synthetic peptide fragments which correspond to the produced peptide fragments from the panel; (c) performing selected reaction monitoring mass spectrometry to measure the abundance of the peptide fragments from step (b); (d) calculating a probability of lung cancer score based on the peptide fragment measurements of step (c) and one protein-protein interaction comprising peptide fragments produced from FRIL_HUMAN and COIA1_HUMAN; (e) ruling out lung cancer for the subject if the score in step (d) is lower than a pre-determined score; and (f) performing image monitoring on the subject for at least a one year period if the pulmonary nodule is determined not to be lung cancer.
 2. The method of claim 1, wherein the pulmonary nodule is 30 mm or less.
 3. The method of claim 2, wherein the pulmonary nodule is between 8-30 mm.
 4. The method of claim 1, wherein the subject is a human.
 5. The method of claim 1, wherein the synthetic peptide fragments have an isotopic label attached.
 6. The method of claim 1, wherein a transition for each of the peptide fragments is determined.
 7. The method of claim 1, wherein said score is determined as P_(s)=1/[1+exp(−α−Σ_(i=1) ⁵β_(i)*{hacek over (I)}_(i,s)−γ*{hacek over (I)}_(COIA1)*{hacek over (I)}_(FRIL))], where {hacek over (I)} is Box-Cox transformed and normalized intensity of transition i in said sample (s), βi is the corresponding logistic regression coefficient, α is a panel-specific constant, and γ is a coefficient for the interaction term.
 8. The method of claim 1, wherein the predetermined lung cancer score is obtained from a reference population comprising at least 100 subjects with a lung condition and wherein each subject in the reference population has been assigned a score based on the abundance of peptide fragments produced from at least each of ALDOA_HUMAN, FRIL_HUMAN, LG3BP_HUMAN, TSP1_HUMAN and COIA1_HUMAN obtained from a blood sample.
 9. The method of claim 1, wherein lung cancer is ruled out in a subject if the probability of lung cancer score is 15% or less in comparison to a reference population.
 10. The method of claim 9, wherein lung cancer is ruled out in the subject if the probability of lung cancer score is 10% or less in comparison to a reference population.
 11. The method of claim 10, wherein lung cancer is ruled out in the subject if the probability of lung cancer score is 5% or less in comparison to a reference population.
 12. The method of claim 1, wherein the image monitoring comprises chest computed tomography.
 13. The method of claim 1, wherein at least one step is performed on a computer system.
 14. The method of claim 1, wherein the proteolytic enzyme is trypsin. 